Enhanced placental stem cells and uses thereof

ABSTRACT

Provided herein are placental stem cells that exhibit increased survival (“enhanced placental stem cells”), compositions comprising such placental stem cells, and methods of using such placental stem cells and compositions.

This application is a continuation of U.S. patent application Ser. No.14/774,250, filed Sep. 10, 2015 which is a national stage entry ofInternational Patent Application No. PCT/US2014/025202, filed Mar. 13,2014, which claims priority to U.S. Provisional Patent Application No.61/785,222, filed Mar. 14, 2013, the disclosures of which areincorporated herein by reference in their entireties.

The Sequence Listing for this application was submitted via EFS Web as afile named “16384857.txt” which was created on Feb. 10, 2020. The fileis 1.45 KB. The entire contents of this file is incorporated herein byreference in its entirety.

1. FIELD

Provided herein are placental stem cells that exhibit increased survival(“enhanced placental stem cells”), compositions comprising suchplacental stem cells, and methods of using such placental stem cells andcompositions.

2. BACKGROUND

Because mammalian placentas are plentiful and are normally discarded asmedical waste, they represent a unique source of medically-useful cells,e.g., placental stem cells. When cultured/present in certainenvironments, e.g., in vivo, cells can exhibit decreased survival due tothe presence of environmental factors that act as insults to the cells.There exists a need for populations of placental stem cells that areresistant to such insults, and thus survive for longer periods of timein environments that would normally cause cell survival to diminish.

3. SUMMARY

In one aspect, provided herein is a method of modifying placental stemcells such that the placental stem cells survive for longer durations oftime under certain conditions as compared to corresponding unmodifiedplacental stem cells, e.g., modifying the cells to make them resistantto conditions that lead to cell death. Such modified placental stemcells are herein termed “enhanced placental stem cells.” The enhancedplacental stem cells provided herein demonstrate increased survival, andthus can advantageously be used in therapy, particularly in therapywhere the placental stem cells are exposed to conditions that can leadto cell death, e.g., exposure to host cells, blood and blood components(e.g., serum, antibodies, complement) and other conditions that cause orcontribute to cell death. In another aspect, presented herein areenhanced placental stem cells and pharmaceutical compositions comprisingenhanced placental stem cells.

In certain embodiments, placental stem cells modified in accordance withthe methods described herein are considered enhanced placental stemcells if they are capable of surviving when exposed to a given conditionfor a longer duration of time than corresponding unmodified placentalstem cells exposed to the same condition. For example, in oneembodiment, an enhanced placental stem cell demonstrates increasedsurvival in the presence of serum (e.g., human or rat serum),complement, antibody(ies), other cells (e.g., cells of the immunesystem), or conditions that can lead to anoikis (e.g., low-attachmentconditions) as compared to corresponding unmodified placental stemcells. Corresponding unmodified placental stem cells, as used herein,can include any placental stem cell, or population thereof, having thesame characteristics of the placental stem cell or population thereofused to generate the enhanced placental stem cells to which thecorresponding unmodified placental stem cells are compared to. Forexample, corresponding unmodified placental stem cells can possess anyof the physical and/or morphological characteristics of placental stemcells described in Section 5.3.1, below, and/or can possess any of thecell surface, molecular, and/or genetic markers of placental stem cellsdescribed in Section 5.3.2, below. In certain embodiments, correspondingunmodified placental stem cells, when compared to enhanced placentalstem cells (i.e., placental stem cells modified in accordance with themethods described herein) are the same as the enhanced placental stemcells with respect to physical, morphological, and/or genetic makeupexcept for the fact that the corresponding unmodified placental stemcells have not been modified in accordance with the methods describedherein.

In certain embodiments, placental stem cells modified in accordance withthe methods described herein are considered enhanced placental stemcells if they demonstrate one or more of (i) decreased caspase 3/7activity, (ii) increased mitochondrial membrane potential, and/or (iii)increased metabolic activity when exposed to a given condition ascompared to corresponding unmodified placental stem cells exposed to thesame condition. In a specific embodiment, enhanced placental stem cellsdemonstrate decreased caspase 3/7 activity, increased mitochondrialmembrane potential, and increased metabolic activity when exposed to agiven condition as compared to corresponding unmodified placental stemcells exposed to the same condition. Methods and kits for assayingcaspase 3/7 activity are known in the art, e.g., the Caspase-Glo® 3/7Assay System (Promega). Also known in the art are assays and kits formeasuring cellular metabolic activity (e.g., Cell Titer Glo Kit(Promega), ATP Determination Kit (Life Technologies), and Amplex® RedGlutamic Acid/Glutamate Oxidase Assay Kit (Life Technologies)) andassays and kits for measuring mitochondrial membrane potential (e.g.,TMRE—Mitochondrial Membrane Potential Assay Kit (Abcam). Such methodsand kits also are described herein (see Sections 6.1.1.1.3 and 6.1.1.2,below).

In certain embodiments, placental stem cells modified in accordance withthe methods described herein are considered enhanced placental stemcells if they enter a quiescent state when exposed to a condition knownto cause cell death of the placental stem cell, e.g., culturing of theplacental stem cells in serum (e.g., rat serum).

In one embodiment, provided herein is a method of generating enhancedplacental stem cells, comprising contacting a population of placentalstem cells with an effective amount of oligomeric or polymericmolecules, such that one or more genes associated with survival of theplacental stem cells is inhibited (e.g., downregulated as compared toplacental stem cells that have not been modified, e.g., that have notbeen contacted with said molecules). In another embodiment, providedherein is a method of generating enhanced placental stem cells,comprising contacting a population of placental stem cells with aneffective amount of oligomeric or polymeric molecules, such that one ormore genes associated with survival of the placental stem cells isupregulated (e.g., upregulated as compared to placental stem cells thathave not been modified, e.g., that have not been contacted with saidmolecules). In certain embodiments, said oligomeric or polymericmolecules are nucleic acid molecules, such as modulatory RNA molecules.In specific embodiments, the modulatory RNA molecules are microRNAs,microRNA mimics, small interfering RNAs (siRNAs), antisense RNAs,antisense DNAs, small hairpin RNAs (shRNAs), microRNA-adapted shRNA(shRNAmirs), or any combination thereof.

In certain embodiments, the oligomeric or polymeric molecules, e.g.,modulatory RNA molecules, used in the methods described herein forgenerating enhanced placental stem cells target one or more placentalstem cell genes identified herein as being associated with augmentationof placental stem cell survival under suboptimal conditions (hereintermed “survival-associated genes”). For example, the oligomeric orpolymeric molecules, e.g., modulatory RNA molecules, target said one ormore placental stem cell genes as a result of having an RNA or DNAsequence that is complementary to a nucleic acid or amino acid sequenceof said one or more placental stem cell genes. In certain embodiments,inhibition or upregulation of such survival-associated genes inplacental stem cells results in an increased ability of the placentalstem cells to survive in the presence of one or more conditions thatwould otherwise cause death of the placental stem cells. In certainembodiments, inhibition or upregulation of such survival-associatedgenes in placental stem cells results in the ability of the placentalstem cells to survive in the presence of one or more conditions thatwould otherwise cause death of the placental stem cells for a longerduration of time than corresponding unmodified placental stem cells inthe presence of the same condition(s). In certain embodiments,inhibition or upregulation of such survival-associated genes inplacental stem cells results in (i) decreased caspase 3/7 activity, (ii)increased mitochondrial membrane potential, and/or (iii) increasedmetabolic activity of the placental stem cells when exposed to a givencondition as compared to corresponding unmodified placental stem cellsexposed to the same condition. In certain embodiments, inhibition orupregulation of such survival-associated genes in placental stem cellsresults in entry of the placental stem cells into a quiescent state whenexposed to a condition or conditions known to cause cell death of theplacental stem cell. In a specific embodiment, said one or moresurvival-associated genes targeted in the methods described herein toproduce enhanced placental stem cells comprise one or more of the geneslisted in Table 1, below:

TABLE 1 SURVIVAL-ASSOCIATED GENES (Number in parentheses is the NCBIGENE ID NUMBER) ADAMTS9 ABCF2 (1061) DNAJB4 MYB (4602) RTN4 (57142) ANLN(56999) (11080) (54443) BACE1 (23621) ABHD10 EGFR (1956) NAA15 (80155)SEC24A MAP2K1 (55347) (10802) (5604) BCL2 (596) ACTR1A EIF4E (1977)NAA25 (80018) SHOC2 (8036) CCNF (899) (10121) CAV2 (858) ACVR2A (92)EPT1 NAPG (8774) SLC12A2 CDC14A (85465) (6558) (8556) CD276 (80381) ADSS(159) FGF2 (2247) NOB1 (28987) SLC16A3 CDC25A (9123) (993) CDC42 (998)ALG3 (10195) FNDC3B NOTCH2 SLC25A22 CHEK1 (64778) (4853) (79751) (1111)CDK6 (1021) ARHGDIA GALNT7 PAFAH1B2 SLC38A5 CUL2 (8453) (396) (51809)(5049) (92745) COL3A1 (1281) ARL2 (402) GPAM PDCD4 (27250) SLC7A1 FGFR1(2260) (57678) (6541) COL4A1 (1282) ATG9A (79065) HACE1 PDCD6IP SNX15ITPR1 (3708) (57531) (10015) (29907) COL4A2 (1284) PLAG1 (5324) HARS(3035) PHKB (5257) SPTLC1 KIF23 (9493) (10558) CPEB3 (22849) C9ORF167/HARS2 PISD (23761) SQSTM1 TRIM63 TOR4A (54863) (23438) (8878) (84676)CXXC6/TET1 C9ORF89 HERC6 PLK1 (5347) SRPR (6734) CSHL1 (1444) (80312)(84270) (55008) DIABLO CACNA2D1 HMGA1 PNN (5411) SRPRB WEE1 (7465)(56616) (781) (3159) (58477) DNMT3A CAPRIN1 HSDL2 PNPLA6 TMEM43 MLLT1(1788) (4076) (84263) (10908) (79188) (4298) DNMT3B (3) CCDC109A/ IGF2RPPIF (10105) TNFSF9 MMS19 MCU (90550) (3482) (8744) (64210) FGA (2243)CCND1 (595) IPO4 (79711) SIAH1 (6477) TOMM34 RECK (8434) (10953) IMPDH1(3614) CCND3 (896) ITGA2 PPP2R5C TPM3 (7170) RNASEL (3673) (5527) (6041)INSIG1 (3638) CCNE1 (898) KCNN4 PSAT1 (29968) TPPP3 WT1 (7490) (3783)(51673) KREMEN2 CCNT2 (905) KPNA3 PTCD3 (55037) UBE2V1 YIF1B (79412)(3839) (7335) (90522) LPL (4023) CDC14B (8555) LAMC1 PTGS2 (5743) UBE4AZNF622 (3915) (9354) (90441) MCL1 (4170) CDK5RAP1 LAMTOR3 PURA (5813)UGDH (7358) (51654) (8649) PIK3R1 (5295) CENPJ (55835) LUZP1 RAB9B UTP15(7798) (51209) (84135) PPM1D (8493) CHORDC1 LYPLA2 RAD51C VEGFA (26973)(11313) (5889) (7422) SPARC (6678) CREBL2 (1389) PIAS1 (8554) RARS(6097) WNT3A (89780)

In one embodiment, the modulatory RNA molecules used in the methodsdescribed herein for generating enhanced placental stem cells aremicroRNAs (miRNAs). In a specific embodiment, said miRNAs target one ormore (e.g., a combination) of the genes listed in Table 1, above. Inanother specific embodiment, said miRNAs target at least two, at least3, at least 4, or at least 5 of the genes listed in Table 1, above. Inanother specific embodiment, said miRNAs are double-stranded, whereinone strand of said miRNAs have a sequence at least about 70%, 80%, 90%,95%, 98% or 100% complementary to the sequence of one of the genesidentified in Table 1, above (as identified based on the Gene ID of thegene provided in the table).

In another specific embodiment, a microRNA used to generate enhancedplacental stem cells in accordance with the methods described hereinincludes a microRNA listed in Table 2, below. In another specificembodiment, a combination (two or more) of the microRNA listed in Table2, below, are used to generate enhanced placental stem cells inaccordance with the methods described herein. In another specificembodiment, the microRNA used to generate enhanced placental stem cellsin accordance with the methods described herein is miR-16, miR-29a,miR-424, miR-4305, miR-3142, or miR-613. In another specific embodiment,the microRNA used to generate enhanced placental stem cells inaccordance with the methods described herein are a combination of two ormore of miR-16, miR-29a, miR-424, miR-4305, miR-3142, and/or miR-613. Inanother specific embodiment, the microRNA used to generate enhancedplacental stem cells in accordance with the methods described herein ismiR-16. In another specific embodiment, the microRNA used to generateenhanced placental stem cells in accordance with the methods describedherein is miR-29a. In another specific embodiment, the microRNA used togenerate enhanced placental stem cells in accordance with the methodsdescribed herein is miR-424.

TABLE 2 microRNA hsa-miR-424 hsa-miR-141 hsa-miR-3142 hsa-miR-4310hsa-miR-1826 hsa-miR-1308 hsa-miR-143 hsa-miR-581 hsa-miR-1201hsa-miR-1203 hsa-miR-3158 hsa-miR-1227 hsa-miR-136 hsa-miR-362-5phsa-miR-4305 hsa-miR-1271 hsa-miR-1236 hsa-miR-369-5p hsa-miR-662hsa-miR-613 hsa-miR-126* hsa-miR-3123 hsa-miR-432 hsa-miR-450b-5phsa-miR-432* hsa-miR-611 hsa-miR-591 hsa-miR-631 hsa-miR-3170hsa-miR-548k hsa-miR-16 hsa-miR-199a-5p hsa-miR-521 hsa-miR-301ahsa-miR-514b-5p hsa-miR-29a

In another embodiment, the modulatory RNA molecules used in the methodsdescribed herein for generating enhanced placental stem cells aremicroRNA mimics (miRNA mimics). In a specific embodiment, said miRNAmimics target one or more (e.g., a combination) of the genes listed inTable 1, above. In another specific embodiment, said miRNA mimics arebased on one or more of the microRNAs listed in Table 2, above.

In another specific embodiment, the miRNAs or miRNA mimics used in themethods described herein for generating enhanced placental stem cellstarget the placental stem cell survival-associated gene CCND1. Inanother specific embodiment, the miRNAs or miRNA mimics used in themethods described herein for generating enhanced placental stem cellstarget the placental stem cell survival-associated gene CCND3. Inanother specific embodiment, the miRNAs or miRNA mimics used in themethods described herein for generating enhanced placental stem cellstarget the placental stem cell survival-associated gene CCNE1. Inanother specific embodiment, the miRNAs or miRNA mimics used in themethods described herein for generating enhanced placental stem cellstarget the placental stem cell survival-associated gene CCNF. In anotherspecific embodiment, the miRNAs or miRNA mimics used in the methodsdescribed herein for generating enhanced placental stem cells target theplacental stem cell survival-associated gene CDK6. In another specificembodiment, the miRNAs or miRNA mimics used in the methods describedherein for generating enhanced placental stem cells target the placentalstem cell survival-associated gene PPP2R5C. In another specificembodiment, the miRNAs or miRNA mimics used in the methods describedherein for generating enhanced placental stem cells target the placentalstem cell survival-associated gene CDC25A. In another specificembodiment, the miRNAs or miRNA mimics used in the methods describedherein for generating enhanced placental stem cells target the placentalstem cell survival-associated gene WEE1. In another specific embodiment,the miRNAs or miRNA mimics used in the methods described herein forgenerating enhanced placental stem cells target the placental stem cellsurvival-associated gene CHEK1. In another specific embodiment, themiRNAs or miRNA mimics used in the methods described herein forgenerating enhanced placental stem cells target the placental stem cellsurvival-associated gene MCL1. In another specific embodiment, themiRNAs or miRNA mimics used in the methods described herein forgenerating enhanced placental stem cells target the placental stem cellsurvival-associated gene BCL2. In another specific embodiment, themiRNAs or miRNA mimics used in the methods described herein forgenerating enhanced placental stem cells target the placental stem cellsurvival-associated gene PPMID. In another specific embodiment, themiRNAs or miRNA mimics used in the methods described herein forgenerating enhanced placental stem cells target the placental stem cellsurvival-associated gene HMGA1. In another specific embodiment, themiRNAs or miRNA mimics used in the methods described herein forgenerating enhanced placental stem cells target the placental stem cellsurvival-associated gene AKT3. In another specific embodiment, themiRNAs or miRNA mimics used in the methods described herein forgenerating enhanced placental stem cells target the placental stem cellsurvival-associated gene VEGFA. In another specific embodiment, themiRNAs or miRNA mimics used in the methods described herein forgenerating enhanced placental stem cells target the placental stem cellsurvival-associated gene MYB. In another specific embodiment, the miRNAsor miRNA mimics used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene ITGA2.

In another specific embodiment, the miRNAs or miRNA mimics used in themethods described herein for generating enhanced placental stem cellstarget two, three, or more (i.e., a combination) of the followingplacental stem cell survival-associated genes: CCND1, CCND3, CCNE1,CCNF, CDK6, PPP2R5C, CDC25A, WEE1, CHEK1, MCL1, BCL2, PPMID, HMGA1,AKT3, VEGFA, MYB, and/or ITGA2. In another specific embodiment, themiRNAs or miRNA mimics used in the methods described herein forgenerating enhanced placental stem cells target two, three, or more(i.e., a combination) of the following placental stem cellsurvival-associated genes: CCND1, CCND3, CCNE1, CCNF, CDK6, CDC25A,WEE1, CHEK1, and/or MYB. In another specific embodiment, the miRNAs ormiRNA mimics used in the methods described herein for generatingenhanced placental stem cells target two, three, or more (i.e., acombination) of the following placental stem cell survival-associatedgenes: MCL1, PPMID, HMGA1, AKT3, VEGFA, and/or ITGA2.

In another embodiment, the modulatory RNA molecules used in the methodsdescribed herein for generating enhanced placental stem cells are smallinterfering RNAs (siRNAs). In a specific embodiment, said siRNAs targetone or more of the genes listed in Table 1, above. In another specificembodiment, said siRNAs target at least two, at least 3, at least 4, orat least 5 of the genes listed in Table 1, above. In another specificembodiment, said siRNAs are double-stranded, wherein one strand of saidsiRNAs have a sequence at least about 70%, 80%, 90%, 95%, 98% or 100%complementary to the sequence of one of the genes identified in Table 1,above (as identified based on the Gene ID of the gene provided in thetable).

In another specific embodiment, the siRNAs used in the methods describedherein for generating enhanced placental stem cells target the placentalstem cell survival-associated gene CCND1. In another specificembodiment, the siRNAs used in the methods described herein forgenerating enhanced placental stem cells target the placental stem cellsurvival-associated gene CCND3. In another specific embodiment, thesiRNAs used in the methods described herein for generating enhancedplacental stem cells target the placental stem cell survival-associatedgene CCNE1. In another specific embodiment, the siRNAs used in themethods described herein for generating enhanced placental stem cellstarget the placental stem cell survival-associated gene CCNF. In anotherspecific embodiment, the siRNAs used in the methods described herein forgenerating enhanced placental stem cells target the placental stem cellsurvival-associated gene CDK6. In another specific embodiment, thesiRNAs used in the methods described herein for generating enhancedplacental stem cells target the placental stem cell survival-associatedgene PPP2R5C. In another specific embodiment, the siRNAs used in themethods described herein for generating enhanced placental stem cellstarget the placental stem cell survival-associated gene CDC25A. Inanother specific embodiment, the siRNAs used in the methods describedherein for generating enhanced placental stem cells target the placentalstem cell survival-associated gene WEE1. In another specific embodiment,the siRNAs used in the methods described herein for generating enhancedplacental stem cells target the placental stem cell survival-associatedgene CHEK1. In another specific embodiment, the siRNAs used in themethods described herein for generating enhanced placental stem cellstarget the placental stem cell survival-associated gene MCL1. In anotherspecific embodiment, the siRNAs used in the methods described herein forgenerating enhanced placental stem cells target the placental stem cellsurvival-associated gene BCL2. In another specific embodiment, thesiRNAs used in the methods described herein for generating enhancedplacental stem cells target the placental stem cell survival-associatedgene PPMID. In another specific embodiment, the siRNAs used in themethods described herein for generating enhanced placental stem cellstarget the placental stem cell survival-associated gene HMGA1. Inanother specific embodiment, the siRNAs used in the methods describedherein for generating enhanced placental stem cells target the placentalstem cell survival-associated gene AKT3. In another specific embodiment,the siRNAs used in the methods described herein for generating enhancedplacental stem cells target the placental stem cell survival-associatedgene VEGFA. In another specific embodiment, the siRNAs used in themethods described herein for generating enhanced placental stem cellstarget the placental stem cell survival-associated gene MYB. In anotherspecific embodiment, the siRNAs used in the methods described herein forgenerating enhanced placental stem cells target the placental stem cellsurvival-associated gene ITGA2.

In another specific embodiment, the siRNAs used in the methods describedherein for generating enhanced placental stem cells target two, three,or more (i.e., a combination) of the following placental stem cellsurvival-associated genes: CCND1, CCND3, CCNE1, CCNF, CDK6, PPP2R5C,CDC25A, WEE1, CHEK1, MCL1, BCL2, PPMID, HMGA1, AKT3, VEGFA, MYB, and/orITGA2. In another specific embodiment, the siRNAs used in the methodsdescribed herein for generating enhanced placental stem cells targettwo, three, or more (i.e., a combination) of the following placentalstem cell survival-associated genes: CCND1, CCND3, CCNE1, CCNF, CDK6,CDC25A, WEE1, CHEK1, and/or MYB. In another specific embodiment, thesiRNAs used in the methods described herein for generating enhancedplacental stem cells target two, three, or more (i.e., a combination) ofthe following placental stem cell survival-associated genes: MCL1,PPMID, HMGA1, AKT3, VEGFA, and/or ITGA2.

In another embodiment, the modulatory RNA molecules used in the methodsdescribed herein for generating enhanced placental stem cells are smallhairpin RNAs (shRNAs). In a specific embodiment, said shRNAs target oneor more of the survival-associated genes listed in Table 1, above. Inanother specific embodiment, said shRNAs target at least two, at least3, at least 4, or at least 5 of the genes listed in Table 1, above. Inanother specific embodiment, said shRNAs have a sequence at least about70%, 80%, 90%, 95%, 98% or 100% complementary to the sequence of one ofthe genes identified in Table 1, above (as identified based on the GeneID of the gene provided in the table).

In another specific embodiment, the shRNAs used in the methods describedherein for generating enhanced placental stem cells target the placentalstem cell survival-associated gene CCND1. In another specificembodiment, the shRNAs used in the methods described herein forgenerating enhanced placental stem cells target the placental stem cellsurvival-associated gene CCND3. In another specific embodiment, theshRNAs used in the methods described herein for generating enhancedplacental stem cells target the placental stem cell survival-associatedgene CCNE1. In another specific embodiment, the shRNAs used in themethods described herein for generating enhanced placental stem cellstarget the placental stem cell survival-associated gene CCNF. In anotherspecific embodiment, the shRNAs used in the methods described herein forgenerating enhanced placental stem cells target the placental stem cellsurvival-associated gene CDK6. In another specific embodiment, theshRNAs used in the methods described herein for generating enhancedplacental stem cells target the placental stem cell survival-associatedgene PPP2R5C. In another specific embodiment, the shRNAs used in themethods described herein for generating enhanced placental stem cellstarget the placental stem cell survival-associated gene CDC25A. Inanother specific embodiment, the shRNAs used in the methods describedherein for generating enhanced placental stem cells target the placentalstem cell survival-associated gene WEE1. In another specific embodiment,the shRNAs used in the methods described herein for generating enhancedplacental stem cells target the placental stem cell survival-associatedgene CHEK1. In another specific embodiment, the shRNAs used in themethods described herein for generating enhanced placental stem cellstarget the placental stem cell survival-associated gene MCL1. In anotherspecific embodiment, the shRNAs used in the methods described herein forgenerating enhanced placental stem cells target the placental stem cellsurvival-associated gene BCL2. In another specific embodiment, theshRNAs used in the methods described herein for generating enhancedplacental stem cells target the placental stem cell survival-associatedgene PPMID. In another specific embodiment, the shRNAs used in themethods described herein for generating enhanced placental stem cellstarget the placental stem cell survival-associated gene HMGA1. Inanother specific embodiment, the shRNAs used in the methods describedherein for generating enhanced placental stem cells target the placentalstem cell survival-associated gene AKT3. In another specific embodiment,the shRNAs used in the methods described herein for generating enhancedplacental stem cells target the placental stem cell survival-associatedgene VEGFA. In another specific embodiment, the shRNAs used in themethods described herein for generating enhanced placental stem cellstarget the placental stem cell survival-associated gene MYB. In anotherspecific embodiment, the shRNAs used in the methods described herein forgenerating enhanced placental stem cells target the placental stem cellsurvival-associated gene ITGA2.

In another specific embodiment, the shRNAs used in the methods describedherein for generating enhanced placental stem cells target two, three,or more (i.e., a combination) of the following placental stem cellsurvival-associated genes: CCND1, CCND3, CCNE1, CCNF, CDK6, PPP2R5C,CDC25A, WEE1, CHEK1, MCL1, BCL2, PPMID, HMGA1, AKT3, VEGFA, MYB, and/orITGA2. In another specific embodiment, the shRNAs used in the methodsdescribed herein for generating enhanced placental stem cells targettwo, three, or more (i.e., a combination) of the following placentalstem cell survival-associated genes: CCND1, CCND3, CCNE1, CCNF, CDK6,CDC25A, WEE1, CHEK1, and/or MYB. In another specific embodiment, theshRNAs used in the methods described herein for generating enhancedplacental stem cells target two, three, or more (i.e., a combination) ofthe following placental stem cell survival-associated genes: MCL1,PPMID, HMGA1, AKT3, VEGFA, and/or ITGA2.

In another embodiment, the modulatory RNA molecules used in the methodsdescribed herein for generating enhanced placental stem cells areantisense RNAs. In a specific embodiment, said antisense RNAs target oneor more of the survival-associated genes listed in Table 1, above. Inanother specific embodiment, said antisense RNAs target at least two, atleast 3, at least 4, or at least 5 of the genes listed in Table 1,above. In another specific embodiment, said antisense RNAs have asequence at least about 70%, 80%, 90%, 95%, 98% or 100% complementary tothe sequence of one of the genes identified in Table 1, above (asidentified based on the Gene ID of the gene provided in the table).

In another specific embodiment, the antisense RNAs used in the methodsdescribed herein for generating enhanced placental stem cells target theplacental stem cell survival-associated gene CCND1. In another specificembodiment, the antisense RNAs used in the methods described herein forgenerating enhanced placental stem cells target the placental stem cellsurvival-associated gene CCND3. In another specific embodiment, theantisense RNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene CCNE1. In another specific embodiment, theantisense RNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene CCNF. In another specific embodiment, theantisense RNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene CDK6. In another specific embodiment, theantisense RNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene PPP2R5C. In another specific embodiment, theantisense RNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene CDC25A. In another specific embodiment, theantisense RNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene WEE1. In another specific embodiment, theantisense RNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene CHEK1. In another specific embodiment, theantisense RNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene MCL1. In another specific embodiment, theantisense RNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene BCL2. In another specific embodiment, theantisense RNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene PPMID. In another specific embodiment, theantisense RNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene HMGA1. In another specific embodiment, theantisense RNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene AKT3. In another specific embodiment, theantisense RNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene VEGFA. In another specific embodiment, theantisense RNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene MYB. In another specific embodiment, theantisense RNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene ITGA2.

In another specific embodiment, the antisense RNAs used in the methodsdescribed herein for generating enhanced placental stem cells targettwo, three, or more (i.e., a combination) of the following placentalstem cell survival-associated genes: CCND1, CCND3, CCNE1, CCNF, CDK6,PPP2R5C, CDC25A, WEE1, CHEK1, MCL1, BCL2, PPMID, HMGA1, AKT3, VEGFA,MYB, and/or ITGA2. In another specific embodiment, the antisense RNAsused in the methods described herein for generating enhanced placentalstem cells target two, three, or more (i.e., a combination) of thefollowing placental stem cell survival-associated genes: CCND1, CCND3,CCNE1, CCNF, CDK6, CDC25A, WEE1, CHEK1, and/or MYB. In another specificembodiment, the antisense RNAs used in the methods described herein forgenerating enhanced placental stem cells target two, three, or more(i.e., a combination) of the following placental stem cellsurvival-associated genes: MCL1, PPMID, HMGA1, AKT3, VEGFA, and/orITGA2.

In another embodiment, the oligomeric or polymeric molecules used in themethods described herein for generating enhanced placental stem cellsare antisense DNAs. In a specific embodiment, said antisense DNAs targetone or more of the survival-associated genes listed in Table 1, above.In another specific embodiment, said antisense DNAs target at least two,at least 3, at least 4, or at least 5 of the genes listed in Table 1,above. In another specific embodiment, said antisense DNAs have asequence at least about 70%, 80%, 90%, 95%, 98% or 100% complementary tothe sequence of one of the genes identified in Table 1, above (asidentified based on the Gene ID of the gene provided in the table).

In another specific embodiment, the antisense DNAs used in the methodsdescribed herein for generating enhanced placental stem cells target theplacental stem cell survival-associated gene CCND1. In another specificembodiment, the antisense DNAs used in the methods described herein forgenerating enhanced placental stem cells target the placental stem cellsurvival-associated gene CCND3. In another specific embodiment, theantisense DNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene CCNE1. In another specific embodiment, theantisense DNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene CCNF. In another specific embodiment, theantisense DNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene CDK6. In another specific embodiment, theantisense DNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene PPP2R5C. In another specific embodiment, theantisense DNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene CDC25A. In another specific embodiment, theantisense DNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene WEE1. In another specific embodiment, theantisense DNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene CHEK1. In another specific embodiment, theantisense DNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene MCL1. In another specific embodiment, theantisense DNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene BCL2. In another specific embodiment, theantisense DNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene PPMID. In another specific embodiment, theantisense DNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene HMGA1. In another specific embodiment, theantisense DNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene AKT3. In another specific embodiment, theantisense DNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene VEGFA. In another specific embodiment, theantisense DNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene MYB. In another specific embodiment, theantisense DNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene ITGA2.

In another specific embodiment, the antisense DNAs used in the methodsdescribed herein for generating enhanced placental stem cells targettwo, three, or more (i.e., a combination) of the following placentalstem cell survival-associated genes: CCND1, CCND3, CCNE1, CCNF, CDK6,PPP2R5C, CDC25A, WEE1, CHEK1, MCL1, BCL2, PPMID, HMGA1, AKT3, VEGFA,MYB, and/or ITGA2. In another specific embodiment, the antisense DNAsused in the methods described herein for generating enhanced placentalstem cells target two, three, or more (i.e., a combination) of thefollowing placental stem cell survival-associated genes: CCND1, CCND3,CCNE1, CCNF, CDK6, CDC25A, WEE1, CHEK1, and/or MYB. In another specificembodiment, the antisense DNAs used in the methods described herein forgenerating enhanced placental stem cells target two, three, or more(i.e., a combination) of the following placental stem cellsurvival-associated genes: MCL1, PPMID, HMGA1, AKT3, VEGFA, and/orITGA2.

In another aspect, provided herein are isolated enhanced placental stemcells, and compositions thereof, produced according to the methodsdescribed herein, e.g., placental stem cells that have been modified bycontacting said placental stem cells with an effective amount of one ormore oligomeric or polymeric molecules (e.g., modulatory RNA molecules),such that the placental stem cells survive for longer durations of timeunder certain conditions as compared to corresponding unmodifiedplacental stem cells. Such enhanced placental stem cells demonstrateincreased survival under conditions that lead to cell death (e.g., inenvironments where components in the environment can harm the cells,e.g., in vivo) as compared to, e.g., corresponding unmodified placentalstem cells (e.g., corresponding placental stem cells that have not beencontacted with an effective amount of oligomeric or polymeric molecules(e.g., modulatory RNA molecules)). In certain embodiments, the enhancedplacental stem cells provided herein demonstrate increased survival inthe presence of serum (e.g., human or rat serum), complement,antibody(ies), other cells (e.g., cells of the immune system), orconditions that can lead to anoikis (e.g., low-attachment conditions) ascompared to corresponding unmodified placental stem cells.

In certain embodiments, the enhanced placental stem cells providedherein demonstrate one or more of (i) decreased caspase 3/7 activity,(ii) increased mitochondrial membrane potential, and/or (iii) increasedmetabolic activity when exposed to a given condition as compared tocorresponding unmodified placental stem cells exposed to the samecondition. In a specific embodiment, the enhanced placental stem cellsprovided herein demonstrate decreased caspase 3/7 activity and increasedmitochondrial membrane potential, and increased metabolic activity whenexposed to a given condition as compared to corresponding unmodifiedplacental stem cells exposed to the same condition.

In certain embodiments, the enhanced placental stem cells providedherein enter a quiescent state when exposed to a condition known tocause cell death of the placental stem cell, e.g., culturing of theplacental stem cells in serum (e.g., rat serum).

In one embodiment, the isolated enhanced placental stem cells providedherein express at least one survival-associated gene at a decreasedlevel as compared to the expression of the same survival-associated genein a corresponding unmodified placental stem cell. In a specificembodiment, provided herein is an isolated enhanced placental stem cell,or population thereof, wherein said isolated enhanced placental stemcell expresses at least one survival-associated gene from those listedin Table 1 at a decreased level as compared to the expression of thesame survival-associated gene in a corresponding unmodified placentalstem cell. In another specific embodiment, provided herein is anisolated enhanced placental stem cell, or population thereof, whereinsaid isolated enhanced placental stem cell expresses more than onesurvival-associated gene (e.g., a combination) from those listed inTable 1 at a decreased level as compared to the expression of the samesurvival-associated gene in a corresponding unmodified placental stemcell, e.g., the isolated enhanced placental stem cell expresses, two,three, four, five, six, seven, eight, nine, ten, or greater than tengenes from those listed in Table 1 at a decreased level as compared tothe expression of the same survival-associated gene(s) in acorresponding unmodified placental stem cell.

In another embodiment, the isolated enhanced placental stem cellsprovided herein express at least one survival-associated gene at anincreased level as compared to the expression of the samesurvival-associated gene in a corresponding unmodified placental stemcell. In a specific embodiment, provided herein is an isolated enhancedplacental stem cell, or population thereof, wherein said isolatedenhanced placental stem cell expresses at least one survival-associatedgene from those listed in Table 1 at an increased level as compared tothe expression of the same survival-associated gene in a correspondingunmodified placental stem cell. In another specific embodiment, providedherein is an isolated enhanced placental stem cell, or populationthereof, wherein said isolated enhanced placental stem cell expressesmore than one survival-associated gene (e.g., a combination) from thoselisted in Table 1 at an increased level as compared to the expression ofthe same survival-associated gene in a corresponding unmodifiedplacental stem cell, e.g., the isolated enhanced placental stem cellexpresses, two, three, four, five, six, seven, eight, nine, ten, orgreater than ten genes from those listed in Table 1 at an increasedlevel as compared to the expression of the same survival-associatedgene(s) in a corresponding unmodified placental stem cell.

In another specific embodiment, provided herein is an isolated enhancedplacental stem cell, wherein said enhanced placental stem cell expressesthe survival-associated gene CCND1 at a decreased level as compared tothe expression of the survival-associated gene CCND1 in a correspondingunmodified placental stem cell. In another specific embodiment, providedherein is an isolated enhanced placental stem cell, wherein saidenhanced placental stem cell expresses the survival-associated geneCCND3 at a decreased level as compared to the expression of thesurvival-associated gene CCND3 in a corresponding unmodified placentalstem cell. In another specific embodiment, provided herein is anisolated enhanced placental stem cell, wherein said enhanced placentalstem cell expresses the survival-associated gene CCNE1 at a decreasedlevel as compared to the expression of the survival-associated geneCCNE1 in a corresponding unmodified placental stem cell. In anotherspecific embodiment, provided herein is an isolated enhanced placentalstem cell, wherein said enhanced placental stem cell expresses thesurvival-associated gene CCNF at a decreased level as compared to theexpression of the survival-associated gene CCNF in a correspondingunmodified placental stem cell. In another specific embodiment, providedherein is an isolated enhanced placental stem cell, wherein saidenhanced placental stem cell expresses the survival-associated gene CDK6at a decreased level as compared to the expression of thesurvival-associated gene CDK6 in a corresponding unmodified placentalstem cell. In another specific embodiment, provided herein is anisolated enhanced placental stem cell, wherein said enhanced placentalstem cell expresses the survival-associated gene PPP2R5C at a decreasedlevel as compared to the expression of the survival-associated genePPP2R5C in a corresponding unmodified placental stem cell. In anotherspecific embodiment, provided herein is an isolated enhanced placentalstem cell, wherein said enhanced placental stem cell expresses thesurvival-associated gene CDC25A at a decreased level as compared to theexpression of the survival-associated gene CDC25A in a correspondingunmodified placental stem cell.

In another specific embodiment, provided herein is an isolated enhancedplacental stem cell, wherein said enhanced placental stem cell expressesthe survival-associated gene WEE1 at a decreased level as compared tothe expression of the survival-associated gene WEE1 in a correspondingunmodified placental stem cell. In another specific embodiment, providedherein is an isolated enhanced placental stem cell, wherein saidenhanced placental stem cell expresses the survival-associated geneCHEK1 at a decreased level as compared to the expression of thesurvival-associated gene CHEK1 in a corresponding unmodified placentalstem cell. In another specific embodiment, provided herein is anisolated enhanced placental stem cell, wherein said enhanced placentalstem cell expresses the survival-associated gene MCL1 at a decreasedlevel as compared to the expression of the survival-associated gene MCL1in a corresponding unmodified placental stem cell. In another specificembodiment, provided herein is an isolated enhanced placental stem cell,wherein said enhanced placental stem cell expresses thesurvival-associated gene BCL2 at a decreased level as compared to theexpression of the survival-associated gene BCL2 in a correspondingunmodified placental stem cell. In another specific embodiment, providedherein is an isolated enhanced placental stem cell, wherein saidenhanced placental stem cell expresses the survival-associated genePPMID at a decreased level as compared to the expression of thesurvival-associated gene PPMID in a corresponding unmodified placentalstem cell. In another specific embodiment, provided herein is anisolated enhanced placental stem cell, wherein said enhanced placentalstem cell expresses the survival-associated gene HMGA1 at a decreasedlevel as compared to the expression of the survival-associated geneHMGA1 in a corresponding unmodified placental stem cell. In anotherspecific embodiment, provided herein is an isolated enhanced placentalstem cell, wherein said enhanced placental stem cell expresses thesurvival-associated gene AKT3 at a decreased level as compared to theexpression of the survival-associated gene AKT3 in a correspondingunmodified placental stem cell. In another specific embodiment, providedherein is an isolated enhanced placental stem cell, wherein saidenhanced placental stem cell expresses the survival-associated geneVEGFA at a decreased level as compared to the expression of thesurvival-associated gene VEGFA in a corresponding unmodified placentalstem cell. In another specific embodiment, provided herein is anisolated enhanced placental stem cell, wherein said enhanced placentalstem cell expresses the survival-associated gene MYB at a decreasedlevel as compared to the expression of the survival-associated gene MYBin a corresponding unmodified placental stem cell. In another specificembodiment, provided herein is an isolated enhanced placental stem cell,wherein said enhanced placental stem cell expresses thesurvival-associated gene ITGA2 at a decreased level as compared to theexpression of the survival-associated gene ITGA2 in a correspondingunmodified placental stem cell.

In another specific embodiment, provided herein is an isolated enhancedplacental stem cell, wherein said enhanced placental stem cell expressesthe survival-associated gene PPP2R5C at an increased level as comparedto the expression of the survival-associated gene PPP2R5C in acorresponding unmodified placental stem cell. In another specificembodiment, provided herein is an isolated enhanced placental stem cell,wherein said enhanced placental stem cell expresses thesurvival-associated gene MCL1 at an increased level as compared to theexpression of the survival-associated gene MCL1 in a correspondingunmodified placental stem cell. In another specific embodiment, providedherein is an isolated enhanced placental stem cell, wherein saidenhanced placental stem cell expresses the survival-associated genePPMID at an increased level as compared to the expression of thesurvival-associated gene PPMID in a corresponding unmodified placentalstem cell. In another specific embodiment, provided herein is anisolated enhanced placental stem cell, wherein said enhanced placentalstem cell expresses the survival-associated gene HMGA1 at an increasedlevel as compared to the expression of the survival-associated geneHMGA1 in a corresponding unmodified placental stem cell. In anotherspecific embodiment, provided herein is an isolated enhanced placentalstem cell, wherein said enhanced placental stem cell expresses thesurvival-associated gene AKT3 at an increased level as compared to theexpression of the survival-associated gene AKT3 in a correspondingunmodified placental stem cell. In another specific embodiment, providedherein is an isolated enhanced placental stem cell, wherein saidenhanced placental stem cell expresses the survival-associated geneVEGFA at an increased level as compared to the expression of thesurvival-associated gene VEGFA in a corresponding unmodified placentalstem cell. In another specific embodiment, provided herein is anisolated enhanced placental stem cell, wherein said enhanced placentalstem cell expresses the survival-associated gene ITGA2 at an increasedlevel as compared to the expression of the survival-associated geneITGA2 in a corresponding unmodified placental stem cell. In anotherspecific embodiment, provided herein is an isolated enhanced placentalstem cell, wherein said enhanced placental stem cell expresses thesurvival-associated gene CCND1 at an increased level as compared to theexpression of the survival-associated gene CCND1 in a correspondingunmodified placental stem cell. In another specific embodiment, providedherein is an isolated enhanced placental stem cell, wherein saidenhanced placental stem cell expresses the survival-associated geneCCND3 at an increased level as compared to the expression of thesurvival-associated gene CCND3 in a corresponding unmodified placentalstem cell. In another specific embodiment, provided herein is anisolated enhanced placental stem cell, wherein said enhanced placentalstem cell expresses the survival-associated gene CCNE1 at an increasedlevel as compared to the expression of the survival-associated geneCCNE1 in a corresponding unmodified placental stem cell. In anotherspecific embodiment, provided herein is an isolated enhanced placentalstem cell, wherein said enhanced placental stem cell expresses thesurvival-associated gene CDC25A at an increased level as compared to theexpression of the survival-associated gene CDC25A in a correspondingunmodified placental stem cell. In another specific embodiment, providedherein is an isolated enhanced placental stem cell, wherein saidenhanced placental stem cell expresses the survival-associated gene WEE1at an increased level as compared to the expression of thesurvival-associated gene WEE1 in a corresponding unmodified placentalstem cell.

In another specific embodiment, provided herein is an isolated enhancedplacental stem cell, wherein said enhanced placental stem cell expressesone, two, three, or more (i.e., a combination) of the followingplacental stem cell survival-associated genes at a decreased level ascompared to the expression of the same survival-associated gene(s) in acorresponding unmodified placental stem cell: CCND1, CCND3, CCNE1, CCNF,CDK6, PPP2R5C, CDC25A, WEE1, CHEK1, MCL1, BCL2, PPMID, HMGA1, AKT3,VEGFA, MYB, and/or ITGA2. In another specific embodiment, providedherein is an isolated enhanced placental stem cell, wherein saidenhanced placental stem cell (i) expresses one, two, three, or more ofthe following placental stem cell survival-associated genes at adecreased level as compared to the expression of the samesurvival-associated gene(s) in a corresponding unmodified placental stemcell: CCND1, CCND3, CCNE1, CCNF, CDK6, PPP2R5C, CDC25A, WEE1, CHEK1,MCL1, BCL2, PPMID, HMGA1, AKT3, VEGFA, MYB, and/or ITGA2; and (ii)expresses at least one additional survival-associated gene recited inTable 1 at an increased or a decreased level as compared to theexpression of the same survival-associated gene (s) in a correspondingunmodified placental stem cell. Further provided herein are populationsof cells comprising such enhanced placental stem cells and compositionscomprising such enhanced placental stem cells.

In another specific embodiment, provided herein is an isolated enhancedplacental stem cell, wherein said enhanced placental stem cell expressesone, two, three, or more (i.e., a combination) of the followingplacental stem cell survival-associated genes at an increased level ascompared to the expression of the same survival-associated gene(s) in acorresponding unmodified placental stem cell: CCND1, CCND3, CCNE1,PPP2R5C, CDC25A, WEE1, MCL1, PPMID, HMGA1, AKT3, VEGFA, PPP2R5C, and/orITGA2. In another specific embodiment, provided herein is an isolatedenhanced placental stem cell, wherein said enhanced placental stem cell(i) expresses one, two, three, or more of the following placental stemcell survival-associated genes at an increased level as compared to theexpression of the same survival-associated gene(s) in a correspondingunmodified placental stem cell: CCND1, CCND3, CCNE1, PPP2R5C, CDC25A,WEE1, MCL1, PPMID, HMGA1, AKT3, VEGFA, PPP2R5C, and/or ITGA2; and (ii)expresses at least one additional survival-associated gene recited inTable 1 at an increased or a decreased level as compared to theexpression of the same survival-associated gene (s) in a correspondingunmodified placental stem cell. Further provided herein are populationsof cells comprising such enhanced placental stem cells and compositionscomprising such enhanced placental stem cells.

In a specific embodiment, the enhanced placental stem cells describedherein are CD10⁺, CD34⁻, CD105⁺, and CD200⁺. In another specificembodiment, the enhanced placental stem cells described herein expressCD200 and do not express HLA-G; or express CD73, CD105, and CD200; orexpress CD200 and OCT-4; or express CD73 and CD105 and do not expressHLA-G; or express CD73 and CD105 and facilitate the formation of one ormore embryoid-like bodies in a population of placental cells comprisingsaid stem cell when said population is cultured under conditions thatallow for the formation of an embryoid-like body; or express OCT-4 andfacilitate the formation of one or more embryoid-like bodies in apopulation of placental cells comprising said stem cell when saidpopulation is cultured under conditions that allow for the formation ofan embryoid-like body. In another specific embodiment, the enhancedplacental stem cells described herein are additionally CD90⁺ and CD45⁻.In another specific embodiment, the enhanced placental stem cellsdescribed herein are additionally CD80 and CD86⁻. In yet otherembodiments, the enhanced placental stem cells described herein expressone or more of CD44, CD90, HLA-A,B,C or ABC-p, and/or do not express oneor more of CD45, CD117, CD133, KDR, CD80, CD86, HLA-DR, SSEA3, SSEA4, orCD38. In certain embodiments, the enhanced placental stem cellsdescribed herein suppress the activity of an immune cell, e.g., suppressproliferation of a T cell to a detectably greater degree thancorresponding unmodified placental stem cells (e.g., placental cellsthat have not been contacted with an effective amount of oligomeric orpolymeric molecules (e.g., modulatory RNA molecules)), as determinableby, e.g., a mixed leukocyte reaction assay, regression assay, or bead Tcell assay.

In another aspect, provided herein is a method for modulating an immuneresponse, e.g., modulating the immune response of a subject, e.g., ahuman subject, or modulating an immune response in vitro, comprisingcontacting immune cells with the enhanced placental stem cells describedherein, or a composition thereof. In a specific embodiment, the enhancedplacental stem cells provided herein are capable of modulating an immuneresponse to the same degree as an equivalent amount of correspondingunmodified placental stem cells. In another specific embodiment, theenhanced placental stem cells used in a method for modulating an immuneresponse have been modified by contacting said placental stem cells withan effective amount of one or more oligomeric or polymeric molecules(e.g., modulatory RNA molecules) as described herein. Assays formeasuring the ability of cells (e.g., placental stem cells, includingenhanced placental stem cells) to modulate an immune response are knownin the art (see, e.g., U.S. Pat. No. 7,682,803, the disclosure of whichis herein incorporated by reference in its entirety) and describedherein, e.g., mixed lymphocyte reaction, regression assay.

In another aspect, provided herein is a method for promotingangiogenesis, e.g., promoting angiogenesis in a subject, e.g., a humansubject, comprising administering to said subject the enhanced placentalstem cells described herein, or a composition thereof. In a specificembodiment, the enhanced placental stem cells provided herein arecapable of promoting angiogenesis to the same degree as an equivalentamount of corresponding unmodified placental stem cells. In anotherspecific embodiment, the enhanced placental stem cells used in a methodfor promoting angiogenesis have been modified by contacting saidplacental stem cells with an effective amount of one or more oligomericor polymeric molecules (e.g., modulatory RNA molecules) as describedherein. Assays for measuring the ability of cells (e.g., placental stemcells, including enhanced placental stem cells) to promote angiogenesisare known in the art (see, e.g., U.S. Patent Application Publication No.2011/0250182, the disclosure of which is herein incorporated byreference in its entirety), e.g., assaying the ability of cells topromote tube formation by endothelial cells, assaying the ability ofcells to promote endothelial cell migration and/or proliferation, andassaying the ability of cells to secrete factors that promoteangiogenesis.

3.1 Definitions

As used herein, the term “amount,” when referring to placental stemcells, e.g., enhanced placental stem cells described herein, means aparticular number of placental stem cells (e.g., enhanced placental stemcells).

As used herein, the term “derived” means isolated from or otherwisepurified. For example, placental derived adherent cells are isolatedfrom placenta. The term “derived” encompasses cells that are culturedfrom cells isolated directly from a tissue, e.g., the placenta, andcells cultured or expanded from primary isolates.

As used herein, “immunolocalization” means the detection of a compound,e.g., a cellular marker, using an immune protein, e.g., an antibody orfragment thereof in, for example, flow cytometry, fluorescence-activatedcell sorting, magnetic cell sorting, in situ hybridization,immunohistochemistry, or the like.

As used herein, the term “SH2” refers to an antibody that binds anepitope on the marker CD105. Thus, cells that are referred to as SH2⁺are CD105⁺.

As used herein, the terms “SH3” and SH4” refer to antibodies that bindepitopes present on the marker CD73. Thus, cells that are referred to asSH3⁺ and/or SH4⁺ are CD73⁺.

As used herein, a stem cell is “isolated” if at least 50%, 60%, 70%,80%, 90%, 95%, or at least 99% of the other cells with which the stemcell is naturally associated are removed from the stem cell, e.g.,during collection and/or culture of the stem cell. A population of“isolated” cells means a population of cells that is substantiallyseparated from other cells of the tissue, e.g., placenta, from which thepopulation of cells is derived. In some embodiments, a population of,e.g., stem cells is “isolated” if at least 50%, 60%, 70%, 80%, 90%, 95%,or at least 99% of the cells with which the population of stem cells arenaturally associated are removed from the population of stem cells,e.g., during collection and/or culture of the population of stem cells.

As used herein, the term “placental stem cell” refers to a stem cell orprogenitor cell that is derived from, e.g., isolated from, a mammalianplacenta, regardless of the number of passages after a primary culture,which adheres to a tissue culture substrate (e.g., tissue cultureplastic or a fibronectin-coated tissue culture plate). The term“placental stem cell” as used herein does not, however, refer to atrophoblast, a cytotrophoblast, embryonic germ cell, or embryonic stemcell, as those cells are understood by persons of skill in the art. Theterms “placental stem cell” and “placenta-derived stem cell” may be usedinterchangeably. Unless otherwise noted herein, the term “placental”includes the umbilical cord. The placental stem cells disclosed hereinare, in certain embodiments, multipotent in vitro (that is, the cellsdifferentiate in vitro under differentiating conditions), multipotent invivo (that is, the cells differentiate in vivo), or both.

As used herein, a cell is “positive” for a particular marker when thatmarker is detectable. For example, a placental stem cell is positivefor, e.g., CD73 because CD73 is detectable on placental stem cells in anamount detectably greater than background (in comparison to, e.g., anisotype control or an experimental negative control for any givenassay). A cell is also positive for a marker when that marker can beused to distinguish the cell from at least one other cell type, or canbe used to select or isolate the cell when present or expressed by thecell.

As used herein, the term “stem cell” refers to a cell that retains atleast one attribute of a stem cell, e.g., a marker or gene expressionprofile associated with one or more types of stem cells; the ability toreplicate at least 10-40 times in culture; multipotency, e.g., theability to differentiate, either in vitro, in vivo or both, into cellsof one or more of the three germ layers; the lack of adult (i.e.,differentiated) cell characteristics, or the like.

As used herein, “immunomodulation” and “immunomodulatory” mean causing,or having the capacity to cause, a detectable change in an immuneresponse, and the ability to cause a detectable change in an immuneresponse.

As used herein, “immunosuppression” and “immunosuppressive” meancausing, or having the capacity to cause, a detectable reduction in animmune response, and the ability to cause a detectable suppression of animmune response.

As used herein, the term “oligomeric or polymeric molecule” refers to abiomolecule that is capable of targeting a gene, RNA, or protein ofinterest (e.g., by binding or hybridizing to a region of a gene, RNA, orprotein of interest). A gene, RNA, or protein of interest is “targeted”by an oligomeric or polymeric molecule by virtue of the fact that theoligomeric or polymeric molecule is complementary to the nucleic acid oramino acid sequence of the gene, RNA, or protein of interest (thus, thegene, RNA, or protein of interest is a “target” of the oligomeric orpolymeric molecule). Oligomeric and polymeric molecules include, forexample, oligonucleotides, oligonucleosides, oligonucleotide analogs,oligonucleotide mimetics, oligopeptides or polypeptides, and anycombinations (e.g., chimeric combinations) thereof. As such, thesecompounds may be single-stranded, double-stranded, circular, branched orhave hairpins and can comprise structural elements such as internal orterminal bulges or loops. Oligomeric or polymeric double-strandedmolecules can be two strands hybridized to form double-strandedcompounds or a single strand with sufficient self complementarity toallow for hybridization and formation of a fully or partiallydouble-stranded molecule.

As used herein, the term “modulatory RNA molecule” refers to an RNAmolecule that modulates, (e.g., up-regulates or down-regulates) directlyor indirectly, the expression or activity of the selectable target(s)(e.g., a target gene, RNA, or protein). In certain embodiments, a“modulatory RNA molecule” is a siRNA, microRNA (miRNA), microRNA mimic(miRNA mimic), antisense RNA, shRNA, shRNAmir, or a hybrid or acombination thereof that modulates the expression of the selectabletarget in a host cell. In certain embodiments, the modulatory RNAmolecules provided herein comprise about 1 to about 100, from about 8 toabout 80, 10 to 50, 13 to 80, 13 to 50, 13 to 30, 13 to 24, 18 to 22,18-24, 19 to 23, 20 to 80, 20 to 50, 20 to 30, or 20 to 24 nucleobases(i.e. from about 1 to about 100 linked nucleosides).

As used herein, the phrase “increased survival,” when describing thesurvival of enhanced placental stem cells as compared to correspondingunmodified placental stem cells refers to the ability of the enhancedplacental stem cells to remain viable under conditions that cause thedeath (e.g., by apoptosis) of unmodified placental stem cells. Incertain embodiments, increased survival of the enhanced placental stemcells described herein relative to corresponding unmodified placentalstem cells refers to the ability of the enhanced placental stem cells toexhibit at least a 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold,4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold increase insurvival time when cultured under a given condition(s) relative to anequivalent amount of corresponding unmodified placental stem cellscultured under the same condition(s). In certain embodiments, increasedsurvival of the enhanced placental stem cells described herein relativeto corresponding unmodified placental stem cells refers to the abilityof the enhanced placental stem cells to exhibit at least a 1.5-fold to2.5-fold, a 2-fold to 3-fold, a 2.5-fold to 3.5-fold, a 3-fold to4-fold, a 3.5-fold to 4.5-fold, a 4-fold to 5-fold, a 5-fold to 6-fold,a 6-fold to 7-fold, a 7-fold to 8-fold, an 8-fold to 9-fold, or a 9-foldto 10-fold increase in survival time when cultured under a givencondition(s) relative to an equivalent amount of correspondingunmodified placental stem cells cultured under the same condition(s).Survival of enhanced placental stem cells and unmodified placental stemcells can be assessed using methods known in the art, e.g., trypan blueexclusion assay, fluorescein diacetate uptake assay, propidium iodideuptake assay; thymidine uptake assay, and MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Incertain embodiments, increased survival of the enhanced placental stemcells described herein relative to corresponding unmodified placentalstem cells refers to one or more of (i) decreased caspase 3/7 activity,(ii) increased mitochondrial membrane potential, and/or (iii) increasedmetabolic activity in the placental stem cells when cultured under agiven condition(s) (e.g., a condition that that causes cell death) ascompared to corresponding unmodified placental stem cells cultured underthe same condition(s). In certain embodiments, enhanced placental stemcells exhibit (i) at least a 1.5-fold, 2-fold, 2.5-fold, 3-fold,3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or10-fold decrease in caspase 3/7 activity; (ii) at least a 1.5-fold,2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, or 10-fold increase in mitochondrial membranepotential; and/or (iii) at least a 1.5-fold, 2-fold, 2.5-fold, 3-fold,3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or10-fold increase in metabolic activity when cultured under a givencondition(s) (e.g., a condition that that causes cell death) as comparedto corresponding unmodified placental stem cells cultured under the samecondition(s). In certain embodiments, enhanced placental stem cellsexhibit (i) at least a 1.5-fold to 2.5-fold, a 2-fold to 3-fold, a2.5-fold to 3.5-fold, a 3-fold to 4-fold, a 3.5-fold to 4.5-fold, a4-fold to 5-fold, a 5-fold to 6-fold, a 6-fold to 7-fold, a 7-fold to8-fold, an 8-fold to 9-fold, or a 9-fold to 10-fold decrease in caspase3/7 activity; (ii) at least a 1.5-fold to 2.5-fold, a 2-fold to 3-fold,a 2.5-fold to 3.5-fold, a 3-fold to 4-fold, a 3.5-fold to 4.5-fold, a4-fold to 5-fold, a 5-fold to 6-fold, a 6-fold to 7-fold, a 7-fold to8-fold, an 8-fold to 9-fold, or a 9-fold to 10-fold increase inmitochondrial membrane potential; and/or (iii) at least a 1.5-fold to2.5-fold, a 2-fold to 3-fold, a 2.5-fold to 3.5-fold, a 3-fold to4-fold, a 3.5-fold to 4.5-fold, a 4-fold to 5-fold, a 5-fold to 6-fold,a 6-fold to 7-fold, a 7-fold to 8-fold, an 8-fold to 9-fold, or a 9-foldto 10-fold increase in metabolic activity when cultured under a givencondition(s) (e.g., a condition that that causes cell death) as comparedto corresponding unmodified placental stem cells cultured under the samecondition(s). Caspase 3/7 activity, mitochondrial membrane potential,and metabolic activity can be assessed using methods known in the art,e.g., as described in Sections 6.1.1.1.3 and 6.1.1.2, below.

As used herein, the phrase “decreased level,” when referring to thelevel of expression of a given gene in an enhanced placental stem cellas compared to the expression of the same gene in a correspondingunmodified placental stem cell means that the expression of the gene inthe enhanced placental stem cell is downregulated or inhibited,resulting in, e.g., a reduction in the mRNA transcript produced by thegene and/or the protein resulting from the expression of the gene. Asused herein, the phrase “increased level,” when referring to the levelof expression of a given gene in an enhanced placental stem cell ascompared to the expression of the same gene in a correspondingunmodified placental stem cell means that the expression of the gene inthe enhanced placental stem cell is upregulated, resulting in, e.g., anincrease in the amount of mRNA transcripts produced by the gene and/oran increase in the amount of protein resulting from the expression ofthe gene. Determination of whether or not a given gene is expressed at adecreased level or an increased level can be accomplished by anyart-recognized method for detection of protein production or nucleicacid production by cells, e.g. nucleic acid-based methods, e.g.,northern blot analysis, reverse transcriptase polymerase chain reaction(RT-PCR), real-time PCR, quantitative PCR, and the like. Expression ofproteins can be assessed using antibodies that bind to the protein ofinterest, e.g., in an ELISA, Western blot, sandwich assay, or the like.In certain embodiments, a gene in an enhanced placental stem cell (e.g.,a survival-associated gene) is expressed at a decreased level if itsexpression is decreased by at least a 1.5-fold, 2-fold, 2.5-fold,3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, or 10-fold as compared to the expression of the gene in acorresponding unmodified placental stem cell. In certain embodiments, agene in an enhanced placental stem cell (e.g., a survival-associatedgene) is expressed at a decreased level if its expression is decreasedby at least 1.5-fold to 2.5-fold, 2-fold to 3-fold, 2.5-fold to3.5-fold, 3-fold to 4-fold, 3.5-fold to 4.5-fold, 4-fold to 5-fold,5-fold to 6-fold, 6-fold to 7-fold, 7-fold to 8-fold, 8-fold to 9-fold,or 9-fold to 10-fold as compared to the expression of the gene in acorresponding unmodified placental stem cell. In certain embodiments, agene in an enhanced placental stem cell (e.g., a survival-associatedgene) is expressed at an increased level if its expression is increasedby at least a 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold,4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold as comparedto the expression of the gene in a corresponding unmodified placentalstem cell. In certain embodiments, a gene in an enhanced placental stemcell (e.g., a survival-associated gene) is expressed at an increasedlevel if its expression is increased by at least 1.5-fold to 2.5-fold,2-fold to 3-fold, 2.5-fold to 3.5-fold, 3-fold to 4-fold, 3.5-fold to4.5-fold, 4-fold to 5-fold, 5-fold to 6-fold, 6-fold to 7-fold, 7-foldto 8-fold, 8-fold to 9-fold, or 9-fold to 10-fold as compared to theexpression of the gene in a corresponding unmodified placental stemcell.

As used herein, the term “effective amount” in the context contactingplacental stem cells with oligomeric or polymeric molecules refers tothe amount of oligomeric or polymeric molecules sufficient to produce anenhanced placental stem cell, as described herein.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts caspase 3/7 activity in placental stem cells transfectedwith different microRNAs as compared to caspase 3/7 activity inplacental stem cells transfected with negative controls or cultured inthe presence of a caspase inhibitor.

FIG. 2 depicts metabolic activity of placental stem cells transfectedwith different microRNAs as compared to metabolic activity of placentalstem cells transfected with negative controls.

FIG. 3 depicts mitochondrial membrane potential of placental stem cellstransfected with different microRNAs as compared to mitochondrialmembrane potential of placental stem cells transfected with negativecontrols.

FIG. 4A, FIG. 4B and FIG. 4C depict expression levels of: miR-29A (FIG.4A), miR-16 (FIG. 4B), and miR-424 (FIG. 4C) in placental stem cellstransfected with the different miRs as compared to levels observed whenplacental stem cells were transfected with negative controls.

FIG. 5A, FIG. 5B and FIG. 5C depict the effect of miR-29A, miR-16, andmiR-424 transfection on the cell cycle distribution of placental stemcells. (FIG. 5A) Change in G0/G1 Phase; (FIG. 5B) Change in S Phase;(FIG. 5C) Change in G2/M Phase.

FIG. 6A and FIG. 6B depicts the effect of miR-29A, miR-16, and miR-424on placental stem cell expression of Cyclin D3 (FIG. 6A) and Cyclin E(FIG. 6B).

5. DETAILED DESCRIPTION

5.1 Production of Enhanced Placental Stem Cells

In one aspect, provided herein are methods of modifying placental stemcells such that the placental stem cells survive for longer durations oftime under certain conditions as compared to corresponding unmodifiedplacental stem cells, e.g., to make the placental stem cells resistantto conditions that lead to cell death (i.e., to make enhanced placentalstem cells). Such methods comprise contacting the placental stem cellswith an effective amount of one or more oligomeric or polymericmolecules, such that one or more genes identified herein as beingassociated with survival (“survival-associated genes”) in the placentalstem cells is inhibited, i.e., the expression of the gene in theplacental stem cells contacted with the oligomeric or polymericmolecules is lessened as compared to the expression of the gene incorresponding unmodified placental stem cells. In certain embodiments,the oligomeric or polymeric molecules used in the methods describedherein comprise nucleotides (e.g., DNA or RNA molecules), nucleosides,nucleotide analogs, nucleotide mimetics, polypeptides, nucleotideanalogs, nucleotide mimetics, and any combinations (e.g., chimericcombinations) thereof. In a specific embodiment, the methods describedherein for generating enhanced placental stem cells comprise contactingsaid placental stem cells with a combination of two or more nucleotides(e.g., DNA or RNA molecules), nucleosides, nucleotide analogs,nucleotide mimetics, polypeptides, nucleotide analogs, and/or nucleotidemimetics.

In one embodiment, the nucleotide analog is an RNA analog, for example,an RNA analog that has been modified in the 2′-OH group, e.g. bysubstitution with a group, for example —O—CH₃, —O—CH₂—CH₂—O—CH₃,—O—CH₂—CH₂—CH₂—NH₂, —O—CH₂—CH₂—CH₂—OH or —F.

In certain embodiments, the oligomeric or polymeric molecules used inthe methods described herein comprise one or more modifications (e.g.,chemical modifications) in the sugars, bases, or internucleosidelinkages. As used herein, the term “internucleoside linkage group”refers to a group capable of covalently coupling together twonucleotides, such as between RNA units. Examples include phosphate,phosphodiester groups and phosphorothioate groups. In one embodiment,the oligomeric or polymeric molecules used in the methods describedherein comprise at least one phosphate internucleoside linkage group. Inone embodiment, the oligomeric or polymeric molecules used in themethods described herein comprise at least one phosphodiesterinternucleoside linkage group.

In certain embodiments, the oligomeric or polymeric molecules used inthe methods described herein are single-stranded oligonucleotides orpolynucleotides. In certain embodiments, the oligomeric or polymericmolecules used in the methods described herein are double-strandedoligonucleotides or polynucleotides. In certain embodiments, theoligonucleotides or polynucleotides used in the methods described hereincomprise one or more modifications (e.g., chemical modifications) in thesugars, bases, or internucleoside linkages.

In a specific embodiment, the oligomeric molecules used in the methodsdescribed herein are modulatory RNA molecules. In certain embodiments,the modulatory RNA molecules are microRNAs, small interfering RNAs(siRNAs), antisense RNAs, miR mimics, small hairpin RNAs (shRNAs),microRNA-adapted shRNA (shRNAmirs), or any combination thereof.

In another specific embodiment, the oligomeric molecules used in themethods described herein are antisense DNA molecules.

In another specific embodiment, the methods described herein comprisecontacting placental stem cells with a combination of microRNAs, smallinterfering RNAs (siRNAs), antisense RNAs, antisense DNAs, miR mimics,small hairpin RNAs (shRNAs), and/or microRNA-adapted shRNA (shRNAmirs).

5.1.1 microRNA

In certain embodiments, the methods provided herein for the productionof enhanced placental stem cells comprise contacting placental stemcells with an effective amount of microRNAs or microRNA mimics, suchthat an ability to exhibit increased survival in conditions thatnormally cause cell death in said placental stem cells is conferred,e.g., as compared to placental stem cells that have not been modified,e.g., that have not been contacted with the microRNAs or microRNAmimics. As used herein, the term “microRNA,” “miRNA,” or “miR” refers toshort ribonucleic acid (RNA) molecules, including, but not limited to,mature single stranded miRNAs, precursor miRNAs (pre-miR), and variantsthereof. In some embodiments, the miR inhibitors downregulate (e.g.,inhibit) a target gene by inhibition of one or more endogenous miRs. Inone embodiment, the microRNAs are naturally occurring. In certainembodiments, the microRNAs are post-transcriptional regulators that bindto complementary sequences on target messenger RNA transcripts (mRNAs)and result in translational repression and gene silencing. In certainembodiments, a single precursor miRNA contains more than one maturemiRNA sequence. In other embodiments, multiple precursor miRNAs containthe same mature sequence. Generally, precursor miRNA exists as a hairpinloop structure, with each hairpin flanked by sequences necessary forefficient processing. The precursor thus possesses one strand (“arm”)that results in expressed miRNA and an opposite strand (“arm”). In someembodiments, when the relative abundances clearly indicate which is thepredominantly expressed miRNA, the term “microRNA,” “miRNA,” or “miR”refers to the predominant product, and the term “microRNA*,” “miRNA*,”or “miR*” refers to the opposite arm of the precursor. In oneembodiment, miRNA is the “guide” strand that eventually entersRNA-Induced Silencing Complex (RISC), and miRNA* is the other“passenger” strand. In another embodiment, the level of miRNA* presentin the cell at a lower level (e.g., <15%) relative to the correspondingmiRNA. In some embodiments where there is a higher proportion ofpassenger strand present in the cell, the nomenclature miRNA-3p (i.e.,miRNA derived from the 3′ arm of the precursor miRNA) and miRNA-5p(i.e., miRNA-5p is the miRNA derived from the 5′ arm of the precursormiRNA) is used instead of miRNA/miRNA*.

As used herein, the term “microRNA mimic(s)” or “miR mimic(s)” refers tomolecules that can be used to imitate or mimic the gene silencingability of one or more miRNAs. In one embodiment, the miR mimicsdown-regulate (e.g., inhibit) a target gene by imitating one or moreendogenous miRs. In certain embodiments, miRNA mimics are syntheticnon-coding RNAs (i.e., the miRNA is not obtained by purification from asource of the endogenous miRNA). In certain embodiments, the miRNAmimics are capable of entering the RNAi pathway and regulating geneexpression. In certain embodiments, miRNA mimics can be designed asmature molecules (e.g. single stranded) or mimic precursors (e.g., pri-or pre-miRNAs).

In some embodiments, the microRNAs or miRNA mimics provided hereincomprise nucleic acid (modified or modified nucleic acids) includingoligonucleotides comprising, e.g., RNA, DNA, modified RNA, modified DNA,locked nucleic acids, or 2′-O,4′-C-ethylene-bridged nucleic acids (ENA),or any combination of thereof.

The microRNAs or miR mimics can be single-stranded or double-stranded,and can be modified or unmodified. In certain embodiments, the microRNAsor miR mimics have a length of about 2 to about 30 nucleobases. Incertain embodiments, the microRNAs or miR mimics are single-stranded,and have a length of about 15 to about 30 nucleobases. In someembodiments, the microRNAs are single-stranded, and are about 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleobases inlength.

In a specific embodiment, microRNA that can be used to generate enhancedplacental stem cells in accordance with the methods described herein isa microRNA listed in Table 2, above (or a microRNA mimic thereof). Inanother specific embodiment, more than one of the microRNAs listed inTable 2, above, can be used to generate enhanced placental stem cells inaccordance with the methods described herein. In another specificembodiment, the microRNA used to generate enhanced placental stem cellsin accordance with the methods described herein is miR-16, miR-29a,miR-424, miR-4305, miR-3142, and/or miR-613. In another specificembodiment, the microRNA used to generate enhanced placental stem cellsin accordance with the methods described herein is miR-16. In anotherspecific embodiment, the microRNA used to generate enhanced placentalstem cells in accordance with the methods described herein is miR-29a.In another specific embodiment, the microRNA used to generate enhancedplacental stem cells in accordance with the methods described herein ismiR-424.

In a specific embodiment, provided herein is a method of producingenhanced placental stem cells, comprising contacting a placental stemcell, or population thereof, with one or more microRNAs or miR mimicsthat target one or more genes identified herein as being associated withsurvival in placental stem cells (e.g., one or more of the genesidentified in Table 1, above).

In another specific embodiment, the miRNAs or miRNA mimics used in themethods described herein for generating enhanced placental stem cellstarget the placental stem cell survival-associated gene CCND1. Inanother specific embodiment, the miRNAs or miRNA mimics used in themethods described herein for generating enhanced placental stem cellstarget the placental stem cell survival-associated gene CCND3. Inanother specific embodiment, the miRNAs or miRNA mimics used in themethods described herein for generating enhanced placental stem cellstarget the placental stem cell survival-associated gene CCNE1. Inanother specific embodiment, the miRNAs or miRNA mimics used in themethods described herein for generating enhanced placental stem cellstarget the placental stem cell survival-associated gene CCNF. In anotherspecific embodiment, the miRNAs or miRNA mimics used in the methodsdescribed herein for generating enhanced placental stem cells target theplacental stem cell survival-associated gene CDK6. In another specificembodiment, the miRNAs or miRNA mimics used in the methods describedherein for generating enhanced placental stem cells target the placentalstem cell survival-associated gene PPP2R5C. In another specificembodiment, the miRNAs or miRNA mimics used in the methods describedherein for generating enhanced placental stem cells target the placentalstem cell survival-associated gene CDC25A. In another specificembodiment, the miRNAs or miRNA mimics used in the methods describedherein for generating enhanced placental stem cells target the placentalstem cell survival-associated gene WEE1. In another specific embodiment,the miRNAs or miRNA mimics used in the methods described herein forgenerating enhanced placental stem cells target the placental stem cellsurvival-associated gene CHEK1. In another specific embodiment, themiRNAs or miRNA mimics used in the methods described herein forgenerating enhanced placental stem cells target the placental stem cellsurvival-associated gene MCL1. In another specific embodiment, themiRNAs or miRNA mimics used in the methods described herein forgenerating enhanced placental stem cells target the placental stem cellsurvival-associated gene BCL2. In another specific embodiment, themiRNAs or miRNA mimics used in the methods described herein forgenerating enhanced placental stem cells target the placental stem cellsurvival-associated gene PPMID. In another specific embodiment, themiRNAs or miRNA mimics used in the methods described herein forgenerating enhanced placental stem cells target the placental stem cellsurvival-associated gene HMGA1. In another specific embodiment, themiRNAs or miRNA mimics used in the methods described herein forgenerating enhanced placental stem cells target the placental stem cellsurvival-associated gene AKT3. In another specific embodiment, themiRNAs or miRNA mimics used in the methods described herein forgenerating enhanced placental stem cells target the placental stem cellsurvival-associated gene VEGFA. In another specific embodiment, themiRNAs or miRNA mimics used in the methods described herein forgenerating enhanced placental stem cells target the placental stem cellsurvival-associated gene MYB. In another specific embodiment, the miRNAsor miRNA mimics used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene ITGA2.

In another specific embodiment, the miRNAs or miRNA mimics used in themethods described herein for generating enhanced placental stem cellstarget two, three, or more (i.e., a combination) of the followingplacental stem cell survival-associated genes: CCND1, CCND3, CCNE1,CCNF, CDK6, PPP2R5C, CDC25A, WEE1, CHEK1, MCL1, BCL2, PPMID, HMGA1,AKT3, VEGFA, MYB, and/or ITGA2.

In another specific embodiment, contacting of a survival-associated geneof a placental stem cell (e.g., a gene listed in Table 1, above) withmiRNAs or miRNA mimics results in a decrease in the mRNA level of saidgene in said placental stem cell, e.g., the mRNA level of thesurvival-associated gene in the resulting enhanced placental stem cellsis decreased relative to the mRNA level of the same gene in unmodifiedplacental stem cells (i.e., placental stem cells not contacted with amiRNA or miRNA mimic). In certain embodiments, the mRNA level of asurvival-associated gene in an enhanced placental stem cell producedaccording to the methods described herein is decreased about, up to, orno more than, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, e.g., as compared to theexpression of said gene (mRNA level) in unmodified placental stem cells.

In another specific embodiment, contacting of a survival-associated geneof a placental stem cell (e.g., a gene listed in Table 1, above) withmiRNAs or miRNA mimics results in an increase in the mRNA level of saidgene in said placental stem cell, e.g., the mRNA level of thesurvival-associated gene in the resulting enhanced placental stem cellsis increased relative to the mRNA level of the same gene in unmodifiedplacental stem cells (i.e., placental stem cells not contacted with amiRNA or miRNA mimic). In certain embodiments, the mRNA level of asurvival-associated gene in an enhanced placental stem cell producedaccording to the methods described herein is increased about, up to, orno more than, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, e.g., as compared to theexpression of said gene (mRNA level) in unmodified placental stem cells.

The miRNAs or miRNA mimics used in the methods described herein can besupplied by a commercial vendor (e.g., Ambion; Dharmafect), or can besynthesized by, e.g., solid phase synthesis, or according to theprocedures as described in, e.g., Protocols for Oligonucleotides andAnalogs, Ed. Agrawal (1993), Humana Press; Scaringe, Methods (2001), 23,206-217. Gait et al., Applications of Chemically synthesized RNA in RNA:Protein Interactions, Ed. Smith (1998), 1-36. Gallo et al., Tetrahedron(2001), 57, 5707-5713).

The miRNAs or miRNA mimics used in the methods described herein can beidentified by a variety of methods known in the art. In certainembodiments, such miRNAs or miRNA mimics are identified and obtainedfrom one or more miRNA or miRNA mimic libraries, e.g., a commerciallyavailable library (e.g., Ambion, Anti-miR miRNA Precursor Library HumanV13), optionally by a screening method, e.g., medium or high-throughputscreening. In one embodiment, such a library can encompass a wide rangeof target genes or gene families. The screening method can be carriedout, for example, using automated robotics, liquid handling equipments,data processing software, and/or sensitive detectors, e.g., Precision XSAutomated Pipettor System, EL406 liquid handling system, or synergyplate reader.

5.1.2 siRNAs

In certain embodiments, the methods provided herein for the productionof enhanced placental stem cells comprise contacting placental stemcells with an effective amount of small interfering RNAs (siRNAs), suchthat an ability to exhibit increased survival in conditions thatnormally cause cell death in said placental stem cells is conferred,e.g., as compared to placental stem cells that have not been modified,e.g., that have not been contacted with siRNAs. As used herein, the term“small interfering RNA” or “siRNA” is well known in the art and refersto an RNA molecule that interferes with the expression of a specificgene.

The siRNAs used in the methods described herein can be single-strandedor double-stranded, and can be modified or unmodified. In oneembodiment, the siRNAs used in the methods described herein have one ormore 2′-deoxy or 2′-O-modified bases. In some embodiments, the siRNAsused in the methods described herein have one or more base substitutionsand inversions (e.g., 3-4 nucleobases inversions).

In some embodiments, the siRNAs used in the methods described herein aredouble-stranded. In one embodiment, one strand of the siRNA is antisenseto the target nucleic acid, while the other strand is complementary tothe first strand. In certain embodiments, said siRNAs comprise a centralcomplementary region between the first and second strands and terminalregions that are optionally complementary between said first and secondstrands or with the target RNA.

In certain embodiments, the siRNAs used in the methods described hereinhave a length of about 2 to about 50 nucleobases. In some embodiments,the siRNAs used in the methods described herein are double-stranded, andhave a length of about 5 to 45, about 7 to 40, or about 10 to about 35nucleobases. In some embodiments, the siRNAs used in the methodsdescribed herein are double-stranded, and are about 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, or 35 nucleobases in length.

In certain embodiments, one or both ends of the first and/or secondstrands of the siRNAs used in the methods described herein are modifiedby adding one or more natural or modified nucleobases to form anoverhang. In certain embodiments, one or both ends of the first and/orsecond strands of the siRNAs used in the methods described herein areblunt. It is possible for one end of the first and/or second strands ofthe siRNAs used in the methods described herein to be blunt and theother to have overhanging nucleobases. In one embodiment, said overhangsare about 1 to about 10, about 2 to about 8, about 3 to about 7, about 4to about 6 nucleobase(s) in length. In another embodiment, saidoverhangs are about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleobase(s) inlength. In a specific embodiment, the siRNAs used in the methodsdescribed herein are double-stranded, and have a length of about 21nucleobases. In another specific embodiment, the siRNAs aredouble-stranded, and have a length of about 21 nucleobases comprisingdinucleotide 3′ overhangs (e.g., dinucleotide 3′ DNA overhangs such asUU or TT 3′-overhangs) such that there is a 19 nt complementary regionbetween the sense and anti-sense strands.

In a specific embodiment, provided herein is a method of producingenhanced placental stem cells, comprising contacting a placental stemcell, or population thereof, with one or more siRNAs that target one ormore genes identified herein as being associated with survival inplacental stem cells (e.g., one or more of the genes identified in Table1, above). In one embodiment, said siRNAs are double-stranded. In aspecific embodiment, one strand (e.g., sense strand) of saiddouble-stranded siRNAs has a sequence at least about 70%, 80%, 90%, 95%,98% or 100% complementary to the sequence of one of the genes identifiedin Table 1, above (as identified based on the Gene ID of the geneprovided in the table).

In another specific embodiment, the siRNAs used in the methods describedherein for generating enhanced placental stem cells target the placentalstem cell survival-associated gene CCND1. In another specificembodiment, the siRNAs used in the methods described herein forgenerating enhanced placental stem cells target the placental stem cellsurvival-associated gene CCND3. In another specific embodiment, thesiRNAs used in the methods described herein for generating enhancedplacental stem cells target the placental stem cell survival-associatedgene CCNE1. In another specific embodiment, the siRNAs used in themethods described herein for generating enhanced placental stem cellstarget the placental stem cell survival-associated gene CCNF. In anotherspecific embodiment, the siRNAs used in the methods described herein forgenerating enhanced placental stem cells target the placental stem cellsurvival-associated gene CDK6. In another specific embodiment, thesiRNAs used in the methods described herein for generating enhancedplacental stem cells target the placental stem cell survival-associatedgene PPP2R5C. In another specific embodiment, the siRNAs used in themethods described herein for generating enhanced placental stem cellstarget the placental stem cell survival-associated gene CDC25A. Inanother specific embodiment, the siRNAs used in the methods describedherein for generating enhanced placental stem cells target the placentalstem cell survival-associated gene WEE1. In another specific embodiment,the siRNAs used in the methods described herein for generating enhancedplacental stem cells target the placental stem cell survival-associatedgene CHEK1. In another specific embodiment, the siRNAs used in themethods described herein for generating enhanced placental stem cellstarget the placental stem cell survival-associated gene MCL1. In anotherspecific embodiment, the siRNAs used in the methods described herein forgenerating enhanced placental stem cells target the placental stem cellsurvival-associated gene BCL2. In another specific embodiment, thesiRNAs used in the methods described herein for generating enhancedplacental stem cells target the placental stem cell survival-associatedgene PPMID. In another specific embodiment, the siRNAs used in themethods described herein for generating enhanced placental stem cellstarget the placental stem cell survival-associated gene HMGA1. Inanother specific embodiment, the siRNAs used in the methods describedherein for generating enhanced placental stem cells target the placentalstem cell survival-associated gene AKT3. In another specific embodiment,the siRNAs used in the methods described herein for generating enhancedplacental stem cells target the placental stem cell survival-associatedgene VEGFA. In another specific embodiment, the siRNAs used in themethods described herein for generating enhanced placental stem cellstarget the placental stem cell survival-associated gene MYB. In anotherspecific embodiment, the siRNAs used in the methods described herein forgenerating enhanced placental stem cells target the placental stem cellsurvival-associated gene ITGA2.

In another specific embodiment, the siRNAs used in the methods describedherein for generating enhanced placental stem cells target two, three,or more (i.e., a combination) of the following placental stem cellsurvival-associated genes: CCND1, CCND3, CCNE1, CCNF, CDK6, PPP2R5C,CDC25A, WEE1, CHEK1, MCL1, BCL2, PPMID, HMGA1, AKT3, VEGFA, MYB, and/orITGA2.

In another specific embodiment, contacting of a survival-associated geneof a placental stem cell with siRNAs results in a decrease in the mRNAlevel of said gene in said placental stem cell, e.g., the mRNA level ofthe survival-associated gene in the resulting enhanced placental stemcells is decreased relative to the mRNA level of the same gene inunmodified placental stem cells (i.e., placental stem cells notcontacted with an siRNA). In certain embodiments, the mRNA level of asurvival-associated gene in an enhanced placental stem cell producedaccording to the methods described herein is decreased about, up to, orno more than, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, e.g., as compared to theexpression of said gene (mRNA level) in unmodified placental stem cells.

The siRNAs used in the methods described herein can be supplied by acommercial vendor (e.g., Ambion; Dharmacon), or be synthesized by, e.g.,solid phase synthesis, or according to the procedures as described in,e.g., Protocols for Oligonucleotides and Analogs, Ed. Agrawal (1993),Humana Press; Scaringe, Methods (2001), 23, 206-217. Gait et al.,Applications of Chemically synthesized RNA in RNA: Protein Interactions,Ed. Smith (1998), 1-36. Gallo et al., Tetrahedron (2001), 57,5707-5713).

siRNAs useful for the production of enhanced placental stem cells can beidentified by a variety of methods known in the art. In certainembodiments, such siRNAs are identified and obtained from one or moresiRNA libraries, e.g., a commercially available library (e.g., Ambion,Silencer® Select Human Nuclear Hormone Receptor (HNR) siRNA Library V4;Dharmacon, siRNA library Human ON-TARGETplus siRNA Nuclear ReceptorsSub-Library), optionally by a screening method, e.g., medium orhigh-throughput screening. In one embodiment, such a library canencompass a wide range of genes (e.g., human genome-wide siRNA library),or pre-defined to encompass specific target genes or gene families(e.g., human nuclear receptor siRNA library, phosphatase siRNA library,etc.). The screening method can be carried out, for example, usingautomated robotics, liquid handling equipments, data processingsoftware, and/or sensitive detectors, e.g., Precision XS AutomatedPipettor System, EL406 liquid handling system, or synergy plate reader.

5.1.3 Other Molecules

Other oligomeric or polymeric molecules useful for the production ofenhanced placental stem cells include, for example, antisense RNAs,antisense DNAs, shRNAs, and shRNAmirs. In certain embodiments, thesemolecules can be used in any combination with one another and also canbe used in combination with siRNAs, miR mimics and/or miR inhibitors toproduce the enhanced placental stem cells as described herein.

As used herein, the term “antisense RNA” is an antisense ribonucleicacid molecule. By illustration only and without limitation, antisenseRNAs hybridize to a target nucleic acid (e.g., a gene or mRNA) andmodulate expression activities of the target nucleic acid, such astranscription or translation.

As used herein, the term “antisense DNA” is an antisensedeoxyribonucleic acid molecule. Antisense DNA refers to a DNA sequencethat has a nucleotide sequence complementary to the “sense strand” of agene when read in reverse orientation. By illustration only and withoutlimitation, antisense DNAs hybridize to a target nucleic acid (e.g., agene or mRNA) and modulate expression activities of the target nucleicacid, such as transcription or translation.

As used herein, the term “small hairpin RNA” or “shRNA” refers to an RNAmolecule comprising a stem-loop structure; the term “shRNAmir” refers to“microRNA-adapted shRNA.” In certain embodiments, said shRNA comprises afirst and second region of complementary sequence, the degree ofcomplementarity and orientation of the regions being sufficient suchthat base pairing occurs between the regions, the first and secondregions being joined by a loop region, the loop resulting from a lack ofbase pairing between nucleotides (or nucleotide analogs) within the loopregion. The shRNA hairpin structure can be, for example, cleaved by thecellular machinery into siRNA, which is then bound to the RNA-inducedsilencing complex (RISC). This complex binds to and cleaves mRNAs whichmatch the siRNA that is bound to it.

In some embodiments, shRNAmirs provided herein are shRNA constructs thatmimic naturally occurring primary transcript miRNA with the addition ofan miRNA loop and a miRNA flanking sequence to a shRNA. Without wishingto be bound by any theory, the shRNAmir is first cleaved to produceshRNA by Drosha, and then cleaved again by Dicer to produce siRNA. ThesiRNA is then incorporated into the RISC for target mRNA degradation.This allows the shRNAmir to be cleaved by Drosha thereby allowing for agreater increase in knockdown efficiency. The addition of a miR30 loopand 125 nt of miR30 flanking sequence on either side of the shRNAhairpin has been reported to result in greater than 10-fold increase inDrosha and Dicer processing of the expressed hairpins when compared withconventional shRNA constructs without microRNA.

In a specific embodiment, provided herein is a method of producingenhanced placental stem cells, comprising contacting a placental stemcell, or population thereof, with one or more antisense RNAs, antisenseDNAs, shRNAs, and shRNAmirs that target one or more genessurvival-associated genes, e.g., one or more genes listed in Table 1,above.

In another specific embodiment, the modulatory RNA molecules used in themethods described herein for generating enhanced placental stem cellsare small hairpin RNAs or shRNAs. In a specific embodiment, said shRNAstarget one or more of the survival-associated genes listed in Table 1,above. In another specific embodiment, said shRNAs target at least two,at least 3, at least 4, or at least 5 of the genes listed in Table 1,above. In another specific embodiment, said shRNAs have a sequence atleast about 70%, 80%, 90%, 95%, 98% or 100% complementary to thesequence of one of the genes identified in Table 1, above (as identifiedbased on the Gene ID of the gene provided in the table).

In another specific embodiment, the modulatory RNA molecules used in themethods described herein for generating enhanced placental stem cellsare antisense RNAs. In a specific embodiment, said antisense RNAs targetone or more of the survival-associated genes listed in Table 1, above.In another specific embodiment, said antisense RNAs target at least two,at least 3, at least 4, or at least 5 of the genes listed in Table 1,above. In another specific embodiment, said antisense RNAs have asequence at least about 70%, 80%, 90%, 95%, 98% or 100% complementary tothe sequence of one of the genes identified in Table 1, above (asidentified based on the Gene ID of the gene provided in the table).

In another specific embodiment, the molecules used in the methodsdescribed herein for generating enhanced placental stem cells areantisense DNAs. In a specific embodiment, said antisense DNAs target oneor more of the survival-associated genes listed in Table 1, above. Inanother specific embodiment, said antisense DNAs target at least two, atleast 3, at least 4, or at least 5 of the genes listed in Table 1,above. In another specific embodiment, said antisense DNAs have asequence at least about 70%, 80%, 90%, 95%, 98% or 100% complementary tothe sequence of one of the genes identified in Table 1, above (asidentified based on the Gene ID of the gene provided in the table).

In another specific embodiment, the shRNAs used in the methods describedherein for generating enhanced placental stem cells target the placentalstem cell survival-associated gene CCND1. In another specificembodiment, the shRNAs used in the methods described herein forgenerating enhanced placental stem cells target the placental stem cellsurvival-associated gene CCND3. In another specific embodiment, theshRNAs used in the methods described herein for generating enhancedplacental stem cells target the placental stem cell survival-associatedgene CCNE1. In another specific embodiment, the shRNAs used in themethods described herein for generating enhanced placental stem cellstarget the placental stem cell survival-associated gene CCNF. In anotherspecific embodiment, the shRNAs used in the methods described herein forgenerating enhanced placental stem cells target the placental stem cellsurvival-associated gene CDK6. In another specific embodiment, theshRNAs used in the methods described herein for generating enhancedplacental stem cells target the placental stem cell survival-associatedgene PPP2R5C. In another specific embodiment, the shRNAs used in themethods described herein for generating enhanced placental stem cellstarget the placental stem cell survival-associated gene CDC25A. Inanother specific embodiment, the shRNAs used in the methods describedherein for generating enhanced placental stem cells target the placentalstem cell survival-associated gene WEE1. In another specific embodiment,the shRNAs used in the methods described herein for generating enhancedplacental stem cells target the placental stem cell survival-associatedgene CHEK1. In another specific embodiment, the shRNAs used in themethods described herein for generating enhanced placental stem cellstarget the placental stem cell survival-associated gene MCL1. In anotherspecific embodiment, the shRNAs used in the methods described herein forgenerating enhanced placental stem cells target the placental stem cellsurvival-associated gene BCL2. In another specific embodiment, theshRNAs used in the methods described herein for generating enhancedplacental stem cells target the placental stem cell survival-associatedgene PPMID. In another specific embodiment, the shRNAs used in themethods described herein for generating enhanced placental stem cellstarget the placental stem cell survival-associated gene HMGA1. Inanother specific embodiment, the shRNAs used in the methods describedherein for generating enhanced placental stem cells target the placentalstem cell survival-associated gene AKT3. In another specific embodiment,the shRNAs used in the methods described herein for generating enhancedplacental stem cells target the placental stem cell survival-associatedgene VEGFA. In another specific embodiment, the shRNAs used in themethods described herein for generating enhanced placental stem cellstarget the placental stem cell survival-associated gene MYB. In anotherspecific embodiment, the shRNAs used in the methods described herein forgenerating enhanced placental stem cells target the placental stem cellsurvival-associated gene ITGA2.

In another specific embodiment, the shRNAs used in the methods describedherein for generating enhanced placental stem cells target two, three,or more (i.e., a combination) of the following placental stem cellsurvival-associated genes: CCND1, CCND3, CCNE1, CCNF, CDK6, PPP2R5C,CDC25A, WEE1, CHEK1, MCL1, BCL2, PPMID, HMGA1, AKT3, VEGFA, MYB, and/orITGA2.

In another specific embodiment, the antisense RNAs used in the methodsdescribed herein for generating enhanced placental stem cells target theplacental stem cell survival-associated gene CCND1. In another specificembodiment, the antisense RNAs used in the methods described herein forgenerating enhanced placental stem cells target the placental stem cellsurvival-associated gene CCND3. In another specific embodiment, theantisense RNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene CCNE1. In another specific embodiment, theantisense RNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene CCNF. In another specific embodiment, theantisense RNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene CDK6. In another specific embodiment, theantisense RNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene PPP2R5C. In another specific embodiment, theantisense RNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene CDC25A. In another specific embodiment, theantisense RNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene WEE1. In another specific embodiment, theantisense RNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene CHEK1. In another specific embodiment, theantisense RNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene MCL1. In another specific embodiment, theantisense RNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene BCL2. In another specific embodiment, theantisense RNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene PPMID. In another specific embodiment, theantisense RNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene HMGA1. In another specific embodiment, theantisense RNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene AKT3. In another specific embodiment, theantisense RNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene VEGFA. In another specific embodiment, theantisense RNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene MYB. In another specific embodiment, theantisense RNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene ITGA2.

In another specific embodiment, the antisense RNAs used in the methodsdescribed herein for generating enhanced placental stem cells targettwo, three, or more (i.e., a combination) of the following placentalstem cell survival-associated genes: CCND1, CCND3, CCNE1, CCNF, CDK6,PPP2R5C, CDC25A, WEE1, CHEK1, MCL1, BCL2, PPMID, HMGA1, AKT3, VEGFA,MYB, and/or ITGA2.

In another specific embodiment, the antisense DNAs used in the methodsdescribed herein for generating enhanced placental stem cells target theplacental stem cell survival-associated gene CCND1. In another specificembodiment, the antisense DNAs used in the methods described herein forgenerating enhanced placental stem cells target the placental stem cellsurvival-associated gene CCND3. In another specific embodiment, theantisense DNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene CCNE1. In another specific embodiment, theantisense DNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene CCNF. In another specific embodiment, theantisense DNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene CDK6. In another specific embodiment, theantisense DNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene PPP2R5C. In another specific embodiment, theantisense DNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene CDC25A. In another specific embodiment, theantisense DNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene WEE1. In another specific embodiment, theantisense DNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene CHEK1. In another specific embodiment, theantisense DNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene MCL1. In another specific embodiment, theantisense DNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene BCL2. In another specific embodiment, theantisense DNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene PPMID. In another specific embodiment, theantisense DNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene HMGA1. In another specific embodiment, theantisense DNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene AKT3. In another specific embodiment, theantisense DNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene VEGFA. In another specific embodiment, theantisense DNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene MYB. In another specific embodiment, theantisense DNAs used in the methods described herein for generatingenhanced placental stem cells target the placental stem cellsurvival-associated gene ITGA2.

In another specific embodiment, the antisense DNAs used in the methodsdescribed herein for generating enhanced placental stem cells targettwo, three, or more (i.e., a combination) of the following placentalstem cell survival-associated genes: CCND1, CCND3, CCNE1, CCNF, CDK6,PPP2R5C, CDC25A, WEE1, CHEK1, MCL1, BCL2, PPMID, HMGA1, AKT3, VEGFA,MYB, and/or ITGA2.

In another specific embodiment, contacting of a survival-associated geneof a placental stem cell with an shRNA or antisense RNA results in adecrease in the mRNA level of said gene in said placental stem cell,e.g., the mRNA level of the survival-associated gene in the resultingenhanced placental stem cells is decreased relative to the mRNA level ofthe same gene in unmodified placental stem cells (i.e., placental stemcells not contacted with an shRNA or antisense RNA). In certainembodiments, the mRNA level of a survival-associated gene in an enhancedplacental stem cell produced according to the methods described hereinis decreased about, up to, or no more than, 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or99%, e.g., as compared to the expression of said gene (mRNA level) inunmodified placental stem cells.

The antisense RNAs, antisense DNAs, shRNAs and shRNAmirs used in themethods described herein can be supplied by a commercial vendor (e.g.,Ambion; Dharmafect), or can be synthesized by, e.g., solid phasesynthesis, or according to the procedures as described in, e.g.,Protocols for Oligonucleotides and Analogs, Ed. Agrawal (1993), HumanaPress; Scaringe, Methods (2001), 23, 206-217. Gait et al., Applicationsof Chemically synthesized RNA in RNA: Protein Interactions, Ed. Smith(1998), 1-36. Gallo et al., Tetrahedron (2001), 57, 5707-5713).

Antisense RNAs, antisense DNAs, shRNAs, shRNAmirs and other moleculesuseful for the production of enhanced placental stem cells can beidentified by a variety of methods known in the art. In certainembodiments, such antisense RNAs, antisense DNAs, shRNAs, shRNAmirs andother modulatory molecules (e.g., modulatory RNA molecules) areidentified and obtained from one or more libraries, e.g., a commerciallyavailable library (Thermo Scientific, shRNAmir libraries), optionally bya screening method, e.g., medium or high-throughput screening. In oneembodiment, such a library can encompass a wide range of genes (e.g.,human genome targeted library), or pre-defined to encompass specifictarget genes or gene families (e.g., human nuclear receptor targetedlibrary, phosphatase targeted library, etc.). The screening method canbe carried out, for example, using automated robotics, liquid handlingequipments, data processing software, and/or sensitive detectors, e.g.,Precision XS Automated Pipettor System, EL406 liquid handling system, orsynergy plate reader.

In certain embodiments, the antisense RNAs, antisense DNAs, shRNAs andshRNAmirs used in the methods described herein comprise about 8 to about100, from about 8 to about 80, 10 to 50, 13 to 80, 13 to 50, 13 to 30,13 to 24, 18 to 22, 19 to 23, 20 to 80, 20 to 50, 20 to 30, or 20 to 24nucleobases (nucleobases (i.e. from about 1 to about 100 linkednucleosides).

The antisense RNAs, antisense DNAs, shRNAs and shRNAmirs used in themethods described herein can be single-stranded or double-stranded,modified or unmodified. In certain embodiments, said antisense RNAs,antisense DNAs, miR mimics, shRNAs, shRNAmirs and other modulatory RNAmolecules comprise about 1 to about 100, from about 8 to about 80, 10 to50, 13 to 80, 13 to 50, 13 to 30, 13 to 24, 18 to 22, 19 to 23, 20 to80, 20 to 50, 20 to 30, or 20 to 24 nucleobases (i.e. from about 1 toabout 100 linked nucleosides). In certain embodiment, the antisenseRNAs, antisense DNAs, shRNAs and shRNAmirs used in the methods describedherein are single-stranded, comprising from about 12 to about 35nucleobases (i.e. from about 12 to about 35 linked nucleosides). In oneembodiment, the antisense RNAs, antisense DNAs, miR mimics, shRNAs andshRNAmirs used in the methods described herein are about 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, or 35 nucleobases in length.

The shRNAmirs used in the methods described herein can be delivered tothe cells by any known method. In a specific embodiment, an shRNAmirused in the methods described herein is incorporated into a eukaryoticexpression vector. In another specific embodiment, an shRNAmir used inthe methods described herein is incorporated into a viral vector forgene expression. Such viral vectors include, but are not limited to,retroviral vectors, e.g., lentivirus, and adenoviruses. In a specificembodiment, an shRNAmir used in the methods described herein isincorporated into a lentiviral vector.

5.1.4 Delivery of Modulatory Molecules to Placental Stem Cells

The modulatory oligomeric or polymeric molecules used in the methodsdescribed herein can be delivered to placental stem cells bytransfection (e.g., transient or stable transfection) or other meansknown in the art. In certain embodiments, said transfection can becarried out, e.g., using lipids (e.g., liposomes), calcium phosphate,cyclodextrin, dendrimers, or polymers (e.g., cationic polymers); byelectroporation, optical transfection, gene electrotransfer,impalefection, gene gun, or magnetofection; via viruses (e.g., viralcarriers); or a combination thereof. In one embodiment, saidtransfection is performed using commercially available transfectionreagents or kits (e.g., Ambion, siPORT™ Amine, siPORT NeoFX's;Dharmafect, Dharmafect 3 Transfection Reagent or Dharmafect 1Transfection Reagent; Invitrogen, Lipofectamine RNAiMAX; Integrated DNATechnologies, Transductin; Minis Bio LLC, TransIT-siQUEST, TransIT-TKO).In a specific embodiment, said transfection can be carried out usingDharmacon ON-TARGET plus SMARTpool® siRNA reagents with the Dharmafect 1Transfection Reagent. In some embodiments, said transfection can be setup in a medium or high-throughput manner, including, but not limited to,use of microtiter plate (e.g., 96-well plate) and microplate reader(e.g., synergy plate reader), or automation system, for example,Precision XS Automated Pipettor System, EL406 liquid handling system. Inother embodiments, said transfection is set up in a large scale,including, but not limited to, the use of tissue culture dishes orculture flasks (e.g., T25, T75, or T225 flasks). Placental stem cellscan be plated and cultured in tissue culture containers, e.g., dishes,flasks, multiwell plates, or the like, for a sufficient time for theplacental stem cells to proliferate to about 20-80% confluence, or about30-70% confluence at the time of transfection. For example, there can beabout 2000, 2500, 3000, 3500, or 4000 placental stem cells per well in a96-well plate at the time of transfection. In one embodiment, placentalstem cells are at about 50% confluence at the time of transfection. Inanother embodiment, there are about 3000 or 3500 placental stem cellsper well in a 96-well plate at the time of transfection. In anotherembodiment, there are about 3500 placental stem cells per well in a96-well plate at the time of transfection.

The modulatory oligomeric or polymeric molecules used in the methodsdescribed herein can, for example, be administered to the cells viatransient or stable transfection. In one embodiment, stable transfectionof modulatory oligomeric or polymeric molecules can be carried out, forexample, by the use of plasmids or expression vectors that expressfunctional modulatory oligomeric or polymeric molecules. In oneembodiment, such plasmids or expression vectors comprise a selectablemarker (e.g., an antibiotic selection marker). In another embodiment,such plasmids or expression vectors comprise a cytomegalovirus (CMV)promoter, an RNA polymerase III (RNA pol III) promoter (e.g., U6 or H1),or an RNA polymerase II (RNA pol II) promoter.

In certain embodiments, the plasmids or expression vectors used inaccordance with the methods described herein are commercially available(e.g., Ambion, pSilencer™ 4.1-CMV vector). Other examples of mammalianexpression vectors include pLOC (Open Biosystems), which contains acytomegalovirus promoter; pCDM8 (Seed, Nature 329:840 (1987)) and pMT2PC(Kaufman et al., EMBO J. 6:187-195 (1987)). Other example expressionvectors that may be used include pFN10A (ACT) FLEXI® Vector (Promega),pFN11A (BIND) FLEXI® Vector (Promega), pGL4.31[luc2P/GAL4UAS/Hygro](Promega), pFC14K (HALOTAG® 7) MCV FLEXI® Vector (Promega), pFC15A(HALOTAG® 7) MCV FLEXI® Vector (Promega), and the like.

When used in mammalian cells, an expression vector's control functionscan be provided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma virus, adenovirus 2, cytomegalovirus,and simian virus 40. Additional suitable expression systems aredescribed, e.g., in chapters 16 and 17 of Sambrook et al., eds.,Molecular Cloning: A Laboratory Manual, 2nd, ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (1989).

Recombinant expression vectors can include one or more control sequencesthat can be, for example, operably linked to the nucleic acid sequenceencoding the gene to be expressed. Such control sequences are described,for example, in Goeddel, Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (1990). In certainembodiments, the vector includes a control sequence that directsconstitutive expression of the nucleotide sequence in the placental stemcells. In certain other embodiments, said vector comprises a controlsequence that is inducible, e.g., by contact with a chemical agent,e.g., tetracycline.

The modulatory oligomeric or polymeric molecules can be administered tothe cells by any technique known to those of skill in the art, e.g., bydirect transfection. For example, said direct transfection can involvethe step of pre-plating the cells prior to transfection, allowing themto reattach and resume growth for a period of time (e.g., 24 hours)before exposure to transfection complexes. The modulatory oligomeric orpolymeric molecules can also be administered to the cells by reversetransfection. For example, said reverse transfection can involve thestep of adding transfection complexes to the cells while they are insuspension, prior to plating.

In various embodiments, the effects of the modulatory oligomeric orpolymeric molecules on placental stem cells, e.g., downregulation of oneor more survival-associated genes in said placental stem cells so as togenerate enhanced placental stem cells from said placental stem cells,can last for up to, about, or no more than, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hours, or 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, or 28 days, or more. In certain embodiments, theenhanced placental stem cells generated using the methods describedherein are used (e.g., administered to a subject) within no more than 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22 or 23 hours, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days of the timethe enhanced placental stem cells are produced. In certain embodiments,the enhanced placental stem cells generated using the methods describedherein are preserved, e.g., cryopreserved, before use (e.g., beforeadministration to a subject). In certain embodiments, the enhancedplacental stem cells generated using the methods described herein arepreserved, e.g., cryopreserved, then modified in accordance with themethods provided herein, then administered to a subject. In certainembodiments, the effects of the modulatory oligomeric or polymericmolecules on the enhanced placental stem cells are inducible. In certainother embodiments, no, or substantially no, cellular expansion(culturing of the enhanced placental stem cells, proliferation, etc.) isperformed between the time the placental stem cells are modified toproduce the enhanced placental stem cells and the time the enhancedplacental stem cells are administered or cryopreserved.

Assessment of the function (e.g., silencing of survival-associatedgenes) of the modulatory oligomeric or polymeric molecules used in themethods described herein, e.g., the level or degree of gene silencing,can be accomplished by any art-recognized method for detection ofprotein production or nucleic acid production by cells. For example,assessment can be performed by determining the mRNA or protein level ofa gene of interest in a sample of enhanced placental stem cells (e.g., asample of 10×10⁵ to 10×10⁷ enhanced placental stem cells, or 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, or 10% of said enhanced placental stem cells) ascompared to equivalent placental stem cells that have not beentransfected with such a nucleic acid sequence. Such assessment can beperformed using, e.g. nucleic acid-based methods, e.g., northern blotanalysis, reverse transcriptase polymerase chain reaction (RT-PCR),real-time PCR, quantitative PCR, and the like. In other embodiments,expression of protein can be assessed using antibodies that bind to theprotein of interest, e.g., in an ELISA, sandwich assay, or the like. Ina specific embodiment, the enhanced placental stem cells generated usingthe methods described herein produce 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% lessof the mRNA of a target gene (e.g., a survival-associated gene) ascompared to corresponding unmodified placental stem cells (e.g., anequivalent amount of corresponding unmodified placental stem cells). Ina specific embodiment, the enhanced placental stem cells generated usingthe methods described herein produce 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% lessof the protein of a target gene (e.g., a survival-associated gene) ascompared to corresponding unmodified placental stem cells (e.g., anequivalent amount of corresponding unmodified placental stem cells).

5.2 Uses of Enhanced Placental Stem Cells

One advantage of the enhanced placental stem cells described herein isthat they maintain functional characteristics of unmodified placentalstem cells (e.g., the placental stem cells described in U.S. Pat. Nos.7,311,904; 7,311,905; 7,468,276 and 8,057,788, the disclosures of whichare hereby incorporated by reference in their entireties), yetdemonstrate increased survival compared to, e.g., unmodified placentalstem cells, when exposed to (or cultured in) conditions that cause celldeath. Accordingly, the enhanced placental stem cells described hereincan be advantageously used in methods that comprise the administrationof placental stem cells to a subject, wherein the placental stem cellsare exposed to environmental insults upon administration to the subject(e.g., the placental stem cells are exposed to other cells, antibodies,blood components (e.g., complement, serum), and other host cellcomponents following administration).

In one embodiment, the enhanced placental stem cells described hereincan be used in methods of treating an individual having or at risk ofdeveloping a disease, disorder or condition caused by, or relating to,an unwanted or harmful immune response, for instance, a disease,disorder or condition having an inflammatory component. In anotherembodiment, provided herein are methods for the modulation, e.g.,suppression, of the activity, e.g., proliferation, of an immune cell, orplurality of immune cells, by contacting the immune cell(s) with aplurality of enhanced placental stem cells (e.g., a compositioncomprising enhanced placental stem cells). In accordance with suchmethods, a therapeutically effective amount of enhanced placental stemcells can be administered to the individual, wherein the administeredenhanced placental stem cells can survive in said individual for greaterperiods of time than, e.g., unmodified placental stem cells administeredin the same fashion.

In a specific embodiment, provided herein is a method of suppressing animmune response comprising contacting a plurality of immune cells with aplurality of enhanced placental stem cells for a time sufficient forsaid enhanced placental stem cells to detectably suppress an immuneresponse, wherein said enhanced placental stem cells detectably suppressT cell proliferation in a mixed lymphocyte reaction (MLR) assay or aregression assay. An “immune cell” in the context of this method meansany cell of the immune system, particularly T cells and NK (naturalkiller) cells. Thus, in various embodiments of the method, enhancedplacental stem cells are contacted with a plurality of immune cells,wherein the plurality of immune cells are, or comprises, a plurality ofT cells (e.g., a plurality of CD3⁺ T cells, CD4⁺ T cells and/or CD8⁺ Tcells) and/or natural killer cells. An “immune response” in the contextof the method can be any response by an immune cell to a stimulusnormally perceived by an immune cell, e.g., a response to the presenceof an antigen. In various embodiments, an immune response can be theproliferation of T cells (e.g., CD3⁺ T cells, CD4⁺ T cells and/or CD8⁺ Tcells) in response to a foreign antigen, such as an antigen present in atransfusion or graft, or to a self-antigen, as in an autoimmune disease.The immune response can also be a proliferation of T cells containedwithin a graft. The immune response can also be any activity of anatural killer (NK) cell, the maturation of a dendritic cell, or thelike. The immune response can also be a local, tissue- ororgan-specific, or systemic effect of an activity of one or more classesof immune cells, e.g., the immune response can be graft versus hostdisease, inflammation, formation of inflammation-related scar tissue, anautoimmune condition (e.g., rheumatoid arthritis, Type I diabetes, lupuserythematosus, etc.). and the like.

“Contacting,” as used herein in such a context, encompasses bringing theplacental stem cells and immune cells together in a single container(e.g., culture dish, flask, vial, etc.) or in vivo, for example, in thesame individual (e.g., mammal, for example, human). In one embodiment,the contacting is for a time sufficient, and with a sufficient number ofenhanced placental stem cells and immune cells, that a change in animmune function of the immune cells is detectable. In certainembodiments, said contacting is sufficient to suppress immune function(e.g., T cell proliferation in response to an antigen) by at least 50%,60%, 70%, 80%, 90% or 95%, compared to the immune function in theabsence of the enhanced placental stem cells. Such suppression in an invivo context can be determined in an in vitro assay (see below); thatis, the degree of suppression in the in vitro assay can be extrapolated,for a particular number of enhanced placental stem cells and a number ofimmune cells in a recipient individual, to a degree of suppression inthe individual.

The ability of enhanced placental stem cells to suppress an immuneresponse can be, e.g., assessed in vitro. In certain embodiments, anenhanced placental stem cell provided herein suppresses an immuneresponse at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% as wellas a corresponding unmodified placental stem cell. In certainembodiments, an enhanced placental stem cell provided herein suppressesan immune response to the same extent as a corresponding unmodifiedplacental stem cell. For example, a plurality of enhanced placental stemcells can be tested in an MLR comprising combining CD4⁺ or CD8⁺ T cells,dendritic cells (DC) and enhanced placental stem cells in a ratio ofabout 10:1:2, wherein the T cells are stained with a dye such as, e.g.,CFSE that partitions into daughter cells, and wherein the T cells areallowed to proliferate for about 6 days. The plurality of enhancedplacental stem cells is immunosuppressive if the T cell proliferation at6 days in the presence of enhanced placental stem cells is detectablyreduced compared to T cell proliferation in the presence of DC andabsence of placental stem cells. Additionally, a control usingunmodified placental stem cells can be run in parallel to demonstratethat the enhanced placental stem cells are more immunosuppressive thanunmodified or untreated placental stem cells. In such an MLR, forexample, enhanced placental stem cells can be either thawed or harvestedfrom culture. About 20,000 enhanced placental stem cells are resuspendedin 100 μl of medium (RPMI 1640, 1 mM HEPES buffer, antibiotics, and 5%pooled human serum), and allowed to attach to the bottom of a well for 2hours. CD4⁺ and/or CD8⁺ T cells are isolated from whole peripheral bloodmononuclear cells Miltenyi magnetic beads. The cells are CFSE stained,and a total of 100,000 T cells (CD4⁺ T cells alone, CD8⁺ T cells alone,or equal amounts of CD4⁺ and CD8⁺ T cells) are added per well. Thevolume in the well is brought to 200 μl, and the MLR is allowed toproceed. A regression assay or BTR assay can be used in similar fashion.

In another aspect, provided herein is a method for promotingangiogenesis. In a specific embodiment, provided herein is a method forpromoting angiogenesis in a subject, e.g., a human subject, comprisingadministering to said subject the enhanced placental stem cellsdescribed herein, or a composition thereof. In certain embodiments, anenhanced placental stem cell provided herein promotes angiogenesis atleast 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% as well as acorresponding unmodified placental stem cell. In certain embodiments, anenhanced placental stem cell provided herein promotes angiogenesis tothe same extent as a corresponding unmodified placental stem cell.Assays for measuring the ability of cells (e.g., placental stem cells,including enhanced placental stem cells) to promote angiogenesis areknown in the art (see, e.g., U.S. Patent Application Publication No.2011/0250182, the disclosure of which is herein incorporated byreference in its entirety), e.g., assaying the ability of cells topromote tube formation by endothelial cells, assaying the ability ofcells to promote endothelial cell migration and/or proliferation, andassaying the ability of cells to secrete factors that promoteangiogenesis.

The enhanced placental stem cells described herein can be administeredwith one or more second types of stem cells, e.g., mesenchymal stemcells from bone marrow. Such second stem cells can be administered to anindividual with said enhanced placental stem cells in a ratio of, e.g.,about 1:10 to about 10:1.

The enhanced placental stem cells described herein can be administeredto an individual in any manner known in the art, e.g., systemically,locally, intravenously, intramuscularly, intraperitoneally,intraocularly, parenterally, intrathecally, or directly into an organ,e.g., pancreas. For in vivo administration, the enhanced placental stemcells can be formulated as a pharmaceutical composition, as describedbelow.

5.3 Enhanced Placental Stem Cells and Enhanced Placental Stem CellPopulations

The enhanced placental stem cells provided herein are produced fromplacental stem cells using the methods described herein. In accordancewith the methods described herein for producing enhanced placental stemcells, the enhanced placental stem cells described herein express one ormore survival-associated genes (as identified herein, e.g., one or moresurvival-associated genes identified in Table 1, above) at a decreasedor increased level as compared to the expression of the samesurvival-associated gene in a corresponding unmodified placental stemcell (i.e., the expression of the one or more survival-associated genesis downregulated).

Placental stem cells can be either fetal or maternal in origin (that is,can have the genotype of either the mother or fetus). Populations ofplacental stem cells, or populations of cells comprising placental stemcells, can comprise placental stem cells that are solely fetal ormaternal in origin, or can comprise a mixed population of placental stemcells of both fetal and maternal origin. The placental stem cells, andpopulations of cells comprising the placental stem cells, can beidentified and selected by, e.g., the morphological, marker, and culturecharacteristics discussed below.

5.3.1 Physical and Morphological Characteristics

The placental stem cells used in the methods described herein forgenerating enhanced placental stem cells, when cultured in primarycultures or in cell culture, adhere to the tissue culture substrate,e.g., tissue culture container surface (e.g., tissue culture plastic).Placental stem cells in culture assume a generally fibroblastoid,stellate appearance, with a number of cytoplasmic processes extendingfrom the central cell body. The placental stem cells used in the methodsfor generating enhanced placental stem cells described herein are,however, morphologically differentiable from fibroblasts cultured underthe same conditions, as the placental stem cells exhibit a greaternumber of such processes than do fibroblasts. Morphologically, placentalstem cells are also differentiable from hematopoietic stem cells, whichgenerally assume a more rounded, or cobblestone, morphology in culture.

The enhanced placental stem cells described herein are thus distinctfrom, e.g., fibroblasts and hematopoietic stem cells. Further, theenhanced placental stem cells described herein are distinct from theplacental stem cells used to generate the enhanced placental stem cells,particularly with respect to the ability of the cells to survive whenexposed to and/or cultured under conditions that cause cell death ofplacental stem cells (i.e., unmodified placental stem cells).

5.3.2 Cell Surface, Molecular and Genetic Markers

As with unmodified placental stem cells, the enhanced placental stemcells described herein express a plurality of markers that can be usedto identify and/or isolate the enhanced placental stem cells, orpopulations of cells that comprise the enhanced placental stem cells.Generally, the identifying markers associated with the enhancedplacental stem cells described herein are the same as those that can beused to identify the placental stem cells from which the enhancedplacental stem cells are derived (i.e., the placental stem cells used inthe methods described herein for generating enhanced placental stemcells). Thus, the enhanced placental stem cells described herein arecomparable to unmodified to placental stem cells in terms of cellsurface, molecular, and genetic markers, with the difference between thecells being that the enhanced placental stem cells described hereinexpress at least one of survival-associated gene (e.g., at least one ofthe genes identified in Table 1, above) at a lower level relative to theexpression of said gene in an equivalent amount of correspondingunmodified placental stem cells, i.e., at least one survival-associatedgene is downregulated/inhibited in the enhanced placental stem cellsdescribed herein, wherein said survival-associated gene is notdownregulated/inhibited in unmodified placental stem cells.

The enhanced placental stem cells described herein, like the placentalstem cells from which the enhanced placental stem cells are derived, arenot bone marrow-derived mesenchymal cells, adipose-derived mesenchymalstem cells, or mesenchymal cells obtained from umbilical cord blood,placental blood, or peripheral blood.

In certain embodiments, the enhanced placental stem cells describedherein (and/or the placental stem cells used in the methods describedherein for producing enhanced placental stem cells) are CD34⁻, CD10⁺ andCD105⁺ as detected by flow cytometry. In a specific embodiment, theisolated CD34⁻, CD10⁺, CD105⁺ enhanced placental stem cells describedherein (and/or the placental stem cells used in the methods describedherein for producing enhanced placental stem cells) have the potentialto differentiate into cells of a neural phenotype, cells of anosteogenic phenotype, and/or cells of a chondrogenic phenotype. Inanother specific embodiment, the isolated CD34⁻, CD10⁺, CD105⁺ enhancedplacental stem cells described herein (and/or the placental stem cellsused in the methods described herein for producing enhanced placentalstem cells) are additionally CD200⁺. In another specific embodiment, theisolated CD34⁻, CD10⁺, CD105⁺ enhanced placental stem cells describedherein (and/or the placental stem cells used in the methods describedherein for producing enhanced placental stem cells) are additionallyCD45⁻ or CD90⁺. In another specific embodiment, the isolated CD34⁻,CD10⁺, CD105⁺ enhanced placental stem cells described herein (and/or theplacental stem cells used in the methods described herein for producingenhanced placental stem cells) are additionally CD45⁻ and CD90⁺, asdetected by flow cytometry. In another specific embodiment, the isolatedCD34⁻, CD10⁺, CD105⁺, CD200⁺ enhanced placental stem cells describedherein (and/or the placental stem cells used in the methods describedherein for producing enhanced placental stem cells) are additionallyCD90⁺ or CD45⁻, as detected by flow cytometry. In another specificembodiment, the isolated CD34⁻, CD10⁺, CD105⁺, CD200⁺ enhanced placentalstem cells described herein (and/or the placental stem cells used in themethods described herein for producing enhanced placental stem cells)are additionally CD90⁺ and CD45⁻, as detected by flow cytometry, i.e.,the cells are CD34⁻, CD10⁺, CD45⁻, CD90⁺, CD105⁺ and CD200⁺. In anotherspecific embodiment, said CD34⁻, CD10⁺, CD45⁻, CD90⁺, CD105⁺, CD200⁺enhanced placental stem cells described herein (and/or the placentalstem cells used in the methods described herein for producing enhancedplacental stem cells) are additionally CD80⁻ and CD86⁻.

In certain embodiments, the enhanced placental stem cells describedherein (and/or the placental stem cells used in the methods describedherein for producing enhanced placental stem cells) are CD34⁻, CD10⁺,CD105⁺ and CD200⁺, and one or more of CD38⁻, CD45⁻, CD80⁻, CD86⁻,CD133⁻, HLA-DR,DP,DQ⁻, SSEA3⁻, SSEA4⁻, CD29⁺, CD44⁺, CD73⁺, CD90⁺,CD105⁺, HLA-A,B,C⁺, PDL1⁺, ABC-p⁺, and/or OCT-4⁺, as detected by flowcytometry. In other embodiments, any of the CD34⁻, CD10⁺, CD105⁺enhanced placental stem cells described herein (and/or the placentalstem cells used in the methods described herein for producing enhancedplacental stem cells) are additionally one or more of CD29⁺, CD38⁻,CD44⁺, CD54⁺, SH3⁺ or SH4⁺. In another specific embodiment, the enhancedplacental stem cells described herein (and/or the placental stem cellsused in the methods described herein for producing enhanced placentalstem cells) are additionally CD44⁺. In another specific embodiment ofany of the isolated CD34⁻, CD10⁺, CD105⁺ enhanced placental stem cellsdescribed herein (and/or the placental stem cells used in the methodsdescribed herein for producing enhanced placental stem cells) areadditionally one or more of CD117⁻, CD133⁻, KDR⁻ (VEGFR2⁻), HLA-A,B,C⁺,HLA-DP,DQ,DR⁻, or Programmed Death-1 Ligand (PDL1)⁺, or any combinationthereof.

In another embodiment, the CD34⁻, CD10⁺, CD105⁺ enhanced placental stemcells described herein (and/or the placental stem cells used in themethods described herein for producing enhanced placental stem cells)are additionally one or more of CD13⁺, CD29⁺, CD33⁺, CD38⁻, CD44⁺,CD45⁻, CD54⁺, CD62E⁻, CD62L⁻, CD62P⁻, SH3⁺ (CD73⁺), SH4⁺ (CD73⁺), CD80⁻,CD86⁻, CD90⁺, SH2⁺ (CD105⁺), CD106/VCAM⁺, CD117⁻,CD144/VE-cadherin^(low), CD184/CXCR4⁻, CD200⁺, CD133⁻, OCT-4⁺, SSEA3⁻,SSEA4⁻, ABC-p⁺, KDR⁻ (VEGFR2⁻), HLA-A,B,C⁺, HLA-DP,DQ,DR⁻, HLA-G⁻, orProgrammed Death-1 Ligand (PDL1)⁺, or any combination thereof. Inanother embodiment, the CD34⁻, CD10⁺, CD105⁺ enhanced placental stemcells described herein (and/or the placental stem cells used in themethods described herein for producing enhanced placental stem cells)are additionally CD13⁺, CD29⁺, CD33⁺, CD38⁻, CD44⁺, CD45⁻, CD54/ICAM⁺,CD62E⁻, CD62L⁻, CD62P⁻, SH3⁺ (CD73⁺), SH4⁺ (CD73⁺), CD80⁻, CD86⁻, CD90⁺,SH2⁺ (CD105⁺), CD106/VCAM⁺, CD117⁻, CD144/VE-cadherin^(low),CD184/CXCR4⁻, CD200⁺, CD133⁻, OCT-4⁺, SSEA3⁻, SSEA4⁻, ABC-p⁺, KDR⁻(VEGFR2⁻), HLA-A,B,C⁺, HLA-DP,DQ,DR⁻, HLA-G⁻, and Programmed Death-1Ligand (PDL1)⁺.

In another specific embodiment, any of the enhanced placental stem cellsdescribed herein (and/or the placental stem cells used in the methodsdescribed herein for producing enhanced placental stem cells) areadditionally ABC-p⁺, as detected by flow cytometry, or OCT-4⁺ (POU5F1⁺),as determined by reverse-transcriptase polymerase chain reaction(RT-PCR), wherein ABC-p is a placenta-specific ABC transporter protein(also known as breast cancer resistance protein (BCRP) or asmitoxantrone resistance protein (MXR)), and OCT-4 is the Octamer-4protein (POU5F1). In another specific embodiment, any of the enhancedplacental stem cells described herein (and/or the placental stem cellsused in the methods described herein for producing enhanced placentalstem cells) are additionally SSEA3⁻or SSEA4⁻, as determined by flowcytometry, wherein SSEA3 is Stage Specific Embryonic Antigen 3, andSSEA4 is Stage Specific Embryonic Antigen 4. In another specificembodiment, any of the enhanced placental stem cells described herein(and/or the placental stem cells used in the methods described hereinfor producing enhanced placental stem cells) are additionally SSEA3⁻ andSSEA4⁻.

In another specific embodiment, any of the enhanced placental stem cellsdescribed herein (and/or the placental stem cells used in the methodsdescribed herein for producing enhanced placental stem cells) are, orare additionally, one or more of MHC-I⁺ (e.g., HLA-A,B,C⁺), MHC-II⁻(e.g., HLA-DP,DQ,DR⁻) or HLA-G⁻. In another specific embodiment, any ofthe enhanced placental stem cells described herein (and/or the placentalstem cells used in the methods described herein for producing enhancedplacental stem cells) are additionally MHC-I⁺ (e.g., HLA-A,B,C⁺),MHC-II⁻ (e.g., HLA-DP,DQ,DR⁻) and HLA-G⁻.

Also provided herein are populations of the enhanced placental stemcells described herein. In certain embodiments, described herein arepopulations of enhanced placental stem cells comprising the isolatedenhanced placental stem cells described herein, wherein the populationsof cells comprise, e.g., at least 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% isolatedCD10⁺, CD105⁺ and CD34⁻ enhanced placental stem cells; that is, at least10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95% or 98% of cells in said population are isolatedCD10⁺, CD105⁺ and CD34⁻ enhanced placental stem cells. In a specificembodiment, the isolated CD34⁻, CD10⁺, CD105⁺ enhanced placental stemcells are additionally CD200⁺. In another specific embodiment, theisolated CD34⁻, CD10⁺, CD105⁺, CD200⁺ enhanced placental stem cells areadditionally CD90⁺ or CD45⁻, as detected by flow cytometry. In anotherspecific embodiment, the isolated CD34⁻, CD10⁺, CD105⁺, CD200⁺ enhancedplacental stem cells are additionally CD90⁺ and CD45⁻, as detected byflow cytometry. In another specific embodiment, any of the isolatedCD34⁻, CD10⁺, CD105⁺ enhanced placental stem cells described above areadditionally one or more of CD29⁺, CD38⁻, CD44⁺, CD54⁺, SH3⁺ or SH4⁺. Inanother specific embodiment, the isolated CD34⁻, CD10⁺, CD105⁺ enhancedplacental stem cells, or isolated CD34⁻, CD10⁺, CD105⁺, CD200⁺ placentalstem cells, are additionally CD44⁺. In a specific embodiment of any ofthe populations of cells comprising isolated CD34⁻, CD10⁺, CD105⁺enhanced placental stem cells above, the isolated enhanced placentalstem cells are additionally one or more of CD13⁺, CD29⁺, CD33⁺, CD38⁻,CD44⁺, CD45⁻, CD54⁺, CD62E⁻, CD62L⁻, CD62P⁻, SH3⁺ (CD73⁺), SH4⁺ (CD73⁺),CD80⁻, CD86⁻, CD90⁺, SH2⁺ (CD1051, CD106/VCAM⁺, CD117⁻,CD144/VE-cadherin^(low), CD184/CXCR4⁻, CD200⁺, CD133⁻, OCT-4⁺, SSEA3⁻,SSEA4⁻, ABC-p⁺, KDR⁻ (VEGFR2⁻), HLA-A,B,C⁺, HLA-DP,DQ,DR⁻, HLA-G⁻, orProgrammed Death-1 Ligand (PDL1)⁺, or any combination thereof. Inanother specific embodiment, the CD34⁻, CD10⁺, CD105⁺ enhanced placentalstem cells are additionally CD13⁺, CD29⁺, CD33⁺, CD38⁻, CD44⁺, CD45⁻,CD54/ICAM⁺, CD62E⁻, CD62L⁻, CD62P⁻, SH3⁺ (CD73⁺), SH4⁺ (CD73⁺), CD80⁻,CD86⁻, CD90⁺, SH2⁺ (CD105⁺), CD106/VCAM⁺, CD117⁻,CD144/VE-cadherin^(low), CD184/CXCR4⁻, CD200⁺, CD133⁻, OCT-4⁺, SSEA3⁻,SSEA4⁻, ABC-p⁺, KDR⁻ (VEGFR2⁻), HLA-A,B,C⁺, HLA-DP,DQ,DR⁻, HLA-G⁻, andProgrammed Death-1 Ligand (PDL1)⁺.

In certain embodiments, the isolated enhanced placental stem cells insaid population of cells are one or more, or all, of CD10⁺, CD29⁺,CD34⁻, CD38⁻, CD44⁺, CD45⁻, CD54⁺, CD90⁺, SH2⁺, SH3⁺, SH4⁺, SSEA3⁻,SSEA4⁻, OCT-4⁺, and ABC-p⁺, wherein said the placental stem cells usedin the method of generating said isolated enhanced placental stem cellswere obtained by physical and/or enzymatic disruption of placentaltissue. In a specific embodiment, the isolated enhanced placental stemcells are OCT-4⁺ and ABC-p⁺. In another specific embodiment, theisolated enhanced placental stem cells are OCT-4⁺ and CD34⁻, whereinsaid isolated enhanced placental stem cells have at least one of thefollowing characteristics: CD10⁺, CD29⁺, CD44⁺, CD45⁻, CD54⁺, CD90⁺,SH3⁺, SH4⁺, SSEA3⁻, and SSEA4⁻. In another specific embodiment, theisolated enhanced placental stem cells are OCT-4⁺, CD34⁻, CD10⁺, CD29⁺,CD44⁺, CD45⁻, CD54⁺, CD90⁺, SH3⁺, SH4⁺, SSEA3⁻, and SSEA4⁻. In anotherembodiment, the isolated enhanced placental stem cells are OCT-4⁺,CD34⁻, SSEA3⁻, and SSEA4⁻. In another specific embodiment, the isolatedenhanced placental stem cells are OCT-4⁺ and CD34⁻, and is either SH2⁺or SH3⁺. In another specific embodiment, the isolated enhanced placentalstem cells are OCT-4⁺, CD34⁻, SH2⁺, and SH3⁺. In another specificembodiment, the isolated enhanced placental stem cells are OCT-4⁺,CD34⁻, SSEA3⁻, and SSEA4⁻, and are either SH2⁺ or SH3⁺. In anotherspecific embodiment, the isolated enhanced placental stem cells areOCT-4⁺ and CD34⁻, and either SH2⁺ or SH3⁺, and at least one of CD10⁺,CD29⁺, CD44⁺, CD45⁻, CD54⁺, CD90⁺, SSEA3⁻, or SSEA4⁻. In anotherspecific embodiment, the isolated enhanced placental stem cells areOCT-4⁺, CD34⁻, CD10⁺, CD29⁺, CD44⁺, CD45⁻, CD54⁺, CD90⁺, SSEA3⁻, andSSEA4⁻, and either SH2⁺ or SH3⁺.

In another embodiment, the isolated enhanced placental stem cells areSH2⁺, SH3⁺, SH4⁺ and OCT-4⁺. In another specific embodiment, theisolated enhanced placental stem cells are CD10⁺, CD29⁺, CD44⁺, CD54⁺,CD90⁺, CD34⁻, CD45⁻, SSEA3⁻, or SSEA4⁻. In another embodiment, theisolated enhanced placental stem cells are SH2⁺, SH3⁺, SH4⁺, SSEA3⁻ andSSEA4⁻. In another specific embodiment, the isolated enhanced placentalstem cells are SH2⁺, SH3⁺, SH4⁺, SSEA3⁻ and SSEA4⁻, CD10⁺, CD29⁺, CD44⁺,CD54⁺, CD90⁺, OCT-4⁺, CD34⁻ or CD45⁻.

In another embodiment, the isolated enhanced placental stem cellsdescribed herein are CD10⁺, CD29⁺, CD34⁻, CD44⁺, CD45⁻, CD54⁺, CD90⁺,SH2⁺, SH3⁺, and SH4⁺; wherein said isolated enhanced placental stemcells are additionally one or more of OCT-4⁺, SSEA3⁻ or SSEA4⁻.

In certain embodiments, isolated enhanced placental stem cells areCD200⁺ or HLA-G⁻. In a specific embodiment, the isolated enhancedplacental stem cells are CD200⁺ and HLA-G⁻. In another specificembodiment, the isolated enhanced placental stem cells are additionallyCD73⁺ and CD105⁺. In another specific embodiment, the isolated enhancedplacental stem cells are additionally CD34⁻, CD38⁻ or CD45⁻. In anotherspecific embodiment, the isolated enhanced placental stem cells areadditionally CD34⁻, CD38⁻ and CD45⁻. In another specific embodiment,said enhanced placental stem cells are CD34⁻, CD38⁻, CD45⁻, CD73⁺ andCD105⁺. In another specific embodiment, said isolated CD200⁺ or HLA-G⁻enhanced placental stem cells facilitate the formation of embryoid-likebodies in a population of placental cells comprising the isolatedplacental stem cells, under conditions that allow the formation ofembryoid-like bodies. In another specific embodiment, the isolatedenhanced placental stem cells are isolated away from placental cellsthat are not said enhanced placental stem cells. In another specificembodiment, said isolated enhanced placental stem cells are isolatedaway from placental cells that do not display this combination ofmarkers.

In another embodiment, a cell population useful in the methods andcompositions described herein is a population of cells comprising, e.g.,that is enriched for, CD200⁺, HLA-G⁻ enhanced placental stem cells. In aspecific embodiment, said population is a population of placental cells.In various embodiments, at least about 10%, at least about 20%, at leastabout 30%, at least about 40%, at least about 50%, or at least about 60%of cells in said cell population are isolated CD200⁺, HLA-G⁻ enhancedplacental stem cells. Preferably, at least about 70% of cells in saidcell population are isolated CD200⁺, HLA-G⁻ enhanced placental stemcells. More preferably, at least about 90%, 95%, or 99% of said cellsare isolated CD200⁺, HLA-G⁻ enhanced placental stem cells. In a specificembodiment of the cell populations, said isolated CD200⁺, HLA-G⁻enhanced placental stem cells are also CD73⁺ and CD105⁺. In anotherspecific embodiment, said isolated CD200⁺, HLA-G⁻ enhanced placentalstem cells are also CD34⁻, CD38⁻ or CD45⁻. In another specificembodiment, said isolated CD200⁺, HLA-G⁻ enhanced placental stem cellsare also CD34⁻, CD38⁻, CD45⁻, CD73⁺ and CD105⁺. In another embodiment,said cell population produces one or more embryoid-like bodies whencultured under conditions that allow the formation of embryoid-likebodies. In another specific embodiment, said cell population is isolatedaway from placental cells that are not enhanced placental stem cells. Inanother specific embodiment, said isolated CD200⁺, HLA-G⁻ enhancedplacental stem cells are isolated away from placental cells that do notdisplay these markers.

In another embodiment, the isolated enhanced placental stem cellsdescribed herein are CD73⁺, CD105⁺, and CD200⁺. In another specificembodiment, the isolated enhanced placental stem cells are HLA-G⁻. Inanother specific embodiment, the isolated enhanced placental stem cellsare CD34⁻, CD38⁻ or CD45⁻. In another specific embodiment, the isolatedenhanced placental stem cells are CD34⁻, CD38⁻ and CD45⁻. In anotherspecific embodiment, the isolated enhanced placental stem cells areCD34⁻, CD38⁻, CD45⁻, and HLA-G⁻. In another specific embodiment, theisolated CD73⁺, CD105⁺, and CD200⁺ enhanced placental stem cellsfacilitate the formation of one or more embryoid-like bodies in apopulation of placental cells comprising the isolated enhanced placentalstem cells, when the population is cultured under conditions that allowthe formation of embryoid-like bodies. In another specific embodiment,the isolated enhanced placental stem cells are isolated away fromplacental cells that are not the isolated enhanced placental stem cells.In another specific embodiment, the isolated enhanced placental stemcells are isolated away from placental cells that do not display thesemarkers.

In another embodiment, a cell population useful in the methods andcompositions described herein is a population of cells comprising, e.g.,that is enriched for, isolated CD73⁺, CD105⁺, CD200⁺ enhanced placentalstem cells. In various embodiments, at least about 10%, at least about20%, at least about 30%, at least about 40%, at least about 50%, or atleast about 60% of cells in said cell population are isolated CD73⁺,CD105⁺, CD200⁺ enhanced placental stem cells. In another embodiment, atleast about 70% of said cells in said population of cells are isolatedCD73⁺, CD105⁺, CD200⁺ enhanced placental stem cells. In anotherembodiment, at least about 90%, 95% or 99% of cells in said populationof cells are isolated CD73⁺, CD105⁺, CD200⁺ enhanced placental stemcells. In a specific embodiment of said populations, the isolatedenhanced placental stem cells are HLA-G⁻. In another specificembodiment, the isolated enhanced placental stem cells are additionallyCD34⁻, CD38⁻ or CD45⁻. In another specific embodiment, the isolatedenhanced placental stem cells are additionally CD34⁻, CD38⁻ and CD45⁻.In another specific embodiment, the isolated enhanced placental stemcells are additionally CD34⁻, CD38⁻, CD45⁻, and HLA-G⁻. In anotherspecific embodiment, said population of cells produces one or moreembryoid-like bodies when cultured under conditions that allow theformation of embryoid-like bodies. In another specific embodiment, saidpopulation of enhanced placental stem cells is isolated away fromplacental cells that are not enhanced placental stem cells. In anotherspecific embodiment, said population of enhanced placental stem cells isisolated away from placental cells that do not display thesecharacteristics.

In certain other embodiments, the isolated enhanced placental stem cellsare one or more of CD10⁺, CD29⁺, CD34⁻, CD38⁻, CD44⁺, CD45⁻, CD54⁺,CD90⁺, SH2⁺, SH3⁺, SH4⁺, SSEA3-, SSEA4⁻, OCT-4⁺, HLA-G⁻ or ABC-p⁺. In aspecific embodiment, the isolated enhanced placental stem cells areCD10⁺, CD29⁺, CD34⁻, CD38⁻, CD44⁺, CD45⁻, CD54⁺, CD90⁺, SH2⁺, SH3⁺,SH4⁺, SSEA3-, SSEA4⁻, and OCT-4⁺. In another specific embodiment, theisolated enhanced placental stem cells are CD10⁺, CD29⁺, CD34⁻, CD38⁻,CD45⁻, CD54⁺, SH2⁺, SH3⁺, and SH4⁺. In another specific embodiment, theisolated enhanced placental stem cells CD10⁺, CD29⁺, CD34⁻, CD38⁻,CD45⁻, CD54⁺, SH2⁺, SH3⁺, SH4⁺ and OCT-4⁺. In another specificembodiment, the isolated enhanced placental stem cells are CD10⁺, CD29⁺,CD34⁻, CD38⁻, CD44⁺, CD45⁻, CD54⁺, CD90⁺, HLA-G⁻, SH2⁺, SH3⁺, SH4⁺. Inanother specific embodiment, the isolated enhanced placental stem cellsare OCT-4⁺ and ABC-p⁺. In another specific embodiment, the isolatedenhanced placental stem cells are SH2⁺, SH3⁺, SH4⁺ and OCT-4⁺. Inanother embodiment, the isolated enhanced placental stem cells areOCT-4⁺, CD34⁻, SSEA3⁻, and SSEA4⁻. In a specific embodiment, saidisolated OCT-4⁺, CD34⁻, SSEA3⁻, and SSEA4⁻ enhanced placental stem cellsare additionally CD10⁺, CD29⁺, CD34⁻, CD44⁺, CD45⁻, CD54⁺, CD90⁺, SH2⁺,SH3⁺, and SH4⁺. In another embodiment, the isolated enhanced placentalstem cells are OCT-4⁺ and CD34⁻, and either SH3⁺ or SH4⁺. In anotherembodiment, the isolated enhanced placental stem cells are CD34⁻ andeither CD10⁺, CD29⁺, CD44⁺, CD54⁺, CD90⁺, or OCT-4⁺.

In another embodiment, isolated enhanced placental stem cells are CD200⁺and OCT-4⁺. In a specific embodiment, the isolated enhanced placentalstem cells are CD73⁺ and CD105⁺. In another specific embodiment, saidisolated enhanced placental stem cells are HLA-G⁻. In another specificembodiment, said isolated CD200⁺, OCT-4⁺ enhanced placental stem cellsare CD34⁻, CD38⁻ or CD45⁻. In another specific embodiment, said isolatedCD200⁺, OCT-4⁺ enhanced placental stem cells are CD34⁻, CD38⁻ and CD45⁻.In another specific embodiment, said isolated CD200⁺, OCT-4⁺ enhancedplacental stem cells are CD34⁻, CD38⁻, CD45⁻, CD73⁺, CD105⁺ and HLA-G⁻.In another specific embodiment, the isolated CD200⁺, OCT-4⁺ enhancedplacental stem cells facilitate the production of one or moreembryoid-like bodies by a population of placental cells that comprisesthe enhanced placental stem cells, when the population is cultured underconditions that allow the formation of embryoid-like bodies. In anotherspecific embodiment, said isolated CD200⁺, OCT-4⁺ enhanced placentalstem cells are isolated away from placental cells that are not saidenhanced placental stem cells. In another specific embodiment, saidisolated CD200⁺, OCT-4⁺ enhanced placental stem cells are isolated awayfrom placental cells that do not display these characteristics.

In another embodiment, a cell population useful in the methods andcompositions described herein is a population of cells comprising, e.g.,that is enriched for, CD200⁺, OCT-4+ enhanced placental stem cells. Invarious embodiments, at least about 10%, at least about 20%, at leastabout 30%, at least about 40%, at least about 50%, or at least about 60%of cells in said cell population are isolated CD200⁺, OCT-4⁺ enhancedplacental stem cells. In another embodiment, at least about 70% of saidcells are said isolated CD200⁺, OCT-4⁺ enhanced placental stem cells. Inanother embodiment, at least about 80%, 90%, 95%, or 99% of cells insaid cell population are said isolated CD200⁺, OCT-4⁺ enhanced placentalstem cells. In a specific embodiment of the isolated populations, saidisolated CD200⁺, OCT-4⁺ enhanced placental stem cells are additionallyCD73⁺ and CD105⁺. In another specific embodiment, said isolated CD200⁺,OCT-4⁺ enhanced placental stem cells are additionally HLA-G⁻. In anotherspecific embodiment, said isolated CD200⁺, OCT-4⁺ enhanced placentalstem cells are additionally CD34⁻, CD38⁻ and CD45⁻. In another specificembodiment, said isolated CD200⁺, OCT-4⁺ enhanced placental stem cellsare additionally CD34⁻, CD38⁻, CD45⁻, CD73⁺, CD105⁺ and HLA-G⁻. Inanother specific embodiment, the cell population produces one or moreembryoid-like bodies when cultured under conditions that allow theformation of embryoid-like bodies. In another specific embodiment, saidcell population is isolated away from placental cells that are notisolated CD200⁺, OCT-4⁺ enhanced placental stem cells. In anotherspecific embodiment, said cell population is isolated away fromplacental cells that do not display these markers.

In another embodiment, the isolated enhanced placental stem cells usefulin the methods and compositions described herein are CD73⁺, CD105⁺ andHLA-G⁻. In another specific embodiment, the isolated CD73⁺, CD105⁺ andHLA-G⁻ enhanced placental stem cells are additionally CD34⁻, CD38⁻ orCD45⁻. In another specific embodiment, the isolated CD73⁺, CD105⁺,HLA-G⁻ enhanced placental stem cells are additionally CD34⁻, CD38⁻ andCD45⁻. In another specific embodiment, the isolated CD73⁺, CD105⁺,HLA-G⁻ enhanced placental stem cells are additionally OCT-4⁺. In anotherspecific embodiment, the isolated CD73⁺, CD105⁺, HLA-G⁻ enhancedplacental stem cells are additionally CD200⁺. In another specificembodiment, the isolated CD73⁺, CD105⁺, HLA-G⁻ enhanced placental stemcells are additionally CD34⁻, CD38⁻, CD45⁻, OCT-4⁺ and CD200⁺. Inanother specific embodiment, the isolated CD73⁺, CD105⁺, HLA-G⁻ enhancedplacental stem cells facilitate the formation of embryoid-like bodies ina population of placental cells comprising said enhanced placental stemcells, when the population is cultured under conditions that allow theformation of embryoid-like bodies. In another specific embodiment, theisolated CD73⁺, CD105⁺, HLA-G⁻ enhanced placental stem cells areisolated away from placental cells that are not the isolated CD73⁺,CD105⁺, HLA-G⁻ enhanced placental stem cells. In another specificembodiment, said the isolated CD73⁺, CD105⁺, HLA-G⁻ enhanced placentalstem cells are isolated away from placental cells that do not displaythese markers.

In another embodiment, a cell population useful in the methods andcompositions described herein is a population of cells comprising, e.g.,that is enriched for, isolated CD73⁺, CD105⁺ and HLA-G⁻ enhancedplacental stem cells. In various embodiments, at least about 10%, atleast about 20%, at least about 30%, at least about 40%, at least about50%, or at least about 60% of cells in said population of cells areisolated CD73⁺, CD105⁺, HLA-G⁻ enhanced placental stem cells. In anotherembodiment, at least about 70% of cells in said population of cells areisolated CD73⁺, CD105⁺, HLA-G⁻ enhanced placental stem cells. In anotherembodiment, at least about 90%, 95% or 99% of cells in said populationof cells are isolated CD73⁺, CD105⁺, HLA-G⁻ enhanced placental stemcells. In a specific embodiment of the above populations, said isolatedCD73⁺, CD105⁺, HLA-G⁻ enhanced placental stem cells are additionallyCD34⁻, CD38⁻ or CD45⁻. In another specific embodiment, said isolatedCD73⁺, CD105⁺, HLA-G⁻ enhanced placental stem cells are additionallyCD34⁻, CD38⁻ and CD45⁻. In another specific embodiment, said isolatedCD73⁺, CD105⁺, HLA-G⁻ enhanced placental stem cells are additionallyOCT-4⁺. In another specific embodiment, said isolated CD73⁺, CD105⁺,HLA-G⁻ enhanced placental stem cells are additionally CD200⁺. In anotherspecific embodiment, said isolated CD73⁺, CD105⁺, HLA-G⁻ enhancedplacental stem cells are additionally CD34⁻, CD38⁻, CD45⁻, OCT-4⁺ andCD200⁺. In another specific embodiment, said cell population is isolatedaway from placental cells that are not CD73⁺, CD105⁺, HLA-G⁻ enhancedplacental stem cells. In another specific embodiment, said cellpopulation is isolated away from placental cells that do not displaythese markers.

In another embodiment, the isolated enhanced placental stem cells areCD73⁺ and CD105⁺ and facilitate the formation of one or moreembryoid-like bodies in a population of isolated placental cellscomprising said CD73⁺, CD105⁺ cells when said population is culturedunder conditions that allow formation of embryoid-like bodies. Inanother specific embodiment, said isolated CD73⁺, CD105⁺ enhancedplacental stem cells are additionally CD34⁻, CD38⁻ or CD45⁻. In anotherspecific embodiment, said isolated CD73⁺, CD105⁺ enhanced placental stemcells are additionally CD34⁻, CD38⁻ and CD45⁻. In another specificembodiment, said isolated CD73⁺, CD105⁺ enhanced placental stem cellsare additionally OCT-4⁺. In another specific embodiment, said isolatedCD73⁺, CD105⁺ enhanced placental stem cells are additionally OCT-4⁺,CD34⁻, CD38⁻ and CD45⁻. In another specific embodiment, said isolatedCD73⁺, CD105+ enhanced placental stem cells are isolated away fromplacental cells that are not said cells. In another specific embodiment,said isolated CD73⁺, CD105⁺ enhanced placental stem cells are isolatedaway from placental cells that do not display these characteristics.

In another embodiment, a cell population useful in the methods andcompositions described herein is a population of cells comprising, e.g.,that is enriched for, isolated enhanced placental stem cells that areCD73⁺, CD105⁺ and facilitate the formation of one or more embryoid-likebodies in a population of isolated placental cells comprising said cellswhen said population is cultured under conditions that allow formationof embryoid-like bodies. In various embodiments, at least about 10%, atleast about 20%, at least about 30%, at least about 40%, at least about50%, or at least about 60% of cells in said population of cells are saidisolated CD73⁺, CD105⁺ enhanced placental stem cells. In anotherembodiment, at least about 70% of cells in said population of cells aresaid isolated CD73⁺, CD105⁺ enhanced placental stem cells. In anotherembodiment, at least about 90%, 95% or 99% of cells in said populationof cells are said isolated CD73⁺, CD105⁺ enhanced placental stem cells.In a specific embodiment of the above populations, said isolated CD73⁺,CD105⁺ enhanced placental stem cells are additionally CD34⁻, CD38⁻ orCD45⁻. In another specific embodiment, said isolated CD73⁺, CD105⁺enhanced placental stem cells are additionally CD34⁻, CD38⁻ and CD45⁻.In another specific embodiment, said isolated CD73⁺, CD105⁺ enhancedplacental stem cells are additionally OCT-4⁺. In another specificembodiment, said isolated CD73⁺, CD105⁺ enhanced placental stem cellsare additionally CD200⁺. In another specific embodiment, said isolatedCD73⁺, CD105⁺ enhanced placental stem cells are additionally CD34⁻,CD38⁻, CD45⁻, OCT-4⁺ and CD200⁺. In another specific embodiment, saidcell population is isolated away from placental cells that are not saidisolated CD73⁺, CD105⁺ enhanced placental stem cells. In anotherspecific embodiment, said cell population is isolated away fromplacental cells that do not display these markers.

In another embodiment, the isolated enhanced placental stem cells areOCT-4⁺ and facilitate formation of one or more embryoid-like bodies in apopulation of isolated placental cells comprising said enhancedplacental stem cells when said population of cells is cultured underconditions that allow formation of embryoid-like bodies. In a specificembodiment, said isolated OCT-4⁺ enhanced placental stem cells areadditionally CD73⁺ and CD105⁺. In another specific embodiment, saidisolated OCT-4⁺ enhanced placental stem cells are additionally CD34⁻,CD38⁻, or CD45⁻. In another specific embodiment, said isolated OCT-4⁺enhanced placental stem cells are additionally CD200⁺. In anotherspecific embodiment, said isolated OCT-4⁺ enhanced placental stem cellsare additionally CD73⁺, CD105⁺, CD200⁺, CD34⁻, CD38⁻, and CD45⁻. Inanother specific embodiment, said isolated OCT-4⁺ enhanced placentalstem cells are isolated away from placental cells that are not OCT-4⁺enhanced placental stem cells. In another specific embodiment, saidisolated OCT-4⁺ enhanced placental stem cells are isolated away fromplacental cells that do not display these characteristics.

In another embodiment, a cell population useful in the methods andcompositions described herein is a population of cells comprising, e.g.,that is enriched for, isolated enhanced placental stem cells that areOCT-4⁺ and facilitate the formation of one or more embryoid-like bodiesin a population of isolated placental cells comprising said cells whensaid population is cultured under conditions that allow formation ofembryoid-like bodies. In various embodiments, at least about 10%, atleast about 20%, at least about 30%, at least about 40%, at least about50%, or at least about 60% of cells in said population of cells are saidisolated OCT-4⁺ enhanced placental stem cells. In another embodiment, atleast about 70% of cells in said population of cells are said isolatedOCT-4⁺ enhanced placental stem cells. In another embodiment, at leastabout 80%, 90%, 95% or 99% of cells in said population of cells are saidisolated OCT-4⁺ enhanced placental stem cells. In a specific embodimentof the above populations, said isolated OCT-4⁺ enhanced placental stemcells are additionally CD34⁻, CD38⁻ or CD45⁻. In another specificembodiment, said isolated OCT-4⁺ enhanced placental stem cells areadditionally CD34⁻, CD38⁻ and CD45⁻. In another specific embodiment,said isolated OCT-4⁺ enhanced placental stem cells are additionallyCD73⁺ and CD105⁺. In another specific embodiment, said isolated OCT-4⁺enhanced placental stem cells are additionally CD200⁺. In anotherspecific embodiment, said isolated OCT-4⁺ enhanced placental stem cellsare additionally CD73⁺, CD105⁺, CD200⁺, CD34⁻, CD38⁻, and CD45⁻. Inanother specific embodiment, said cell population is isolated away fromplacental cells that are not said enhanced placental stem cells. Inanother specific embodiment, said cell population is isolated away fromplacental cells that do not display these markers.

In another embodiment, the isolated placental stem cells useful in themethods and compositions described herein are isolated HLA-A,B,C⁺,CD45⁻, CD133⁻ and CD34⁻ enhanced placental stem cells. In anotherembodiment, a cell population useful in the methods and compositionsdescribed herein is a population of cells comprising isolated enhancedplacental stem cells, wherein at least about 70%, at least about 80%, atleast about 90%, at least about 95% or at least about 99% of cells insaid population of cells are isolated HLA-A,B,C⁺, CD45⁻, CD133⁻ andCD34⁻ enhanced placental stem cells. In a specific embodiment, saidisolated enhanced placental stem cells or population of isolatedenhanced placental stem cells is isolated away from placental cells thatare not HLA-A,B,C⁺, CD45⁻, CD133⁻ and CD34⁻ enhanced placental stemcells. In another specific embodiment, said isolated enhanced placentalstem cells are non-maternal in origin. In another specific embodiment,said population of isolated enhanced placental stem cells aresubstantially free of maternal components; e.g., at least about 40%,45%, 5-0%, 55%, 60%, 65%, 70%, 75%, 90%, 85%, 90%, 95%, 98% or 99% ofsaid cells in said population of isolated enhanced placental stem cellsare non-maternal in origin.

In another embodiment, the isolated enhanced placental stem cells usefulin the methods and compositions described herein are isolated CD10⁺,CD13⁺, CD33⁺, CD45⁻, CD117⁻ and CD133⁻ enhanced placental stem cells. Inanother embodiment, a cell population useful in the methods andcompositions described herein is a population of cells comprisingisolated enhanced placental stem cells, wherein at least about 70%, atleast about 80%, at least about 90%, at least about 95% or at leastabout 99% of cells in said population of cells are isolated CD10⁺,CD13⁺, CD33⁺, CD45⁻, CD117⁻ and CD133⁻ enhanced placental stem cells. Ina specific embodiment, said isolated enhanced placental stem cells orpopulation of isolated enhanced placental stem cells is isolated awayfrom placental cells that are not said isolated enhanced placental stemcells. In another specific embodiment, said isolated CD10⁺, CD13⁺,CD33⁺, CD45⁻, CD117⁻ and CD133⁻ enhanced placental stem cells arenon-maternal in origin, i.e., have the fetal genotype. In anotherspecific embodiment, at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 90%, 85%, 90%, 95%, 98% or 99% of said cells in said population ofisolated enhanced placental stem cells, are non-maternal in origin. Inanother specific embodiment, said isolated enhanced placental stem cellsor population of isolated enhanced placental stem cells are isolatedaway from placental cells that do not display these characteristics.

In another embodiment, the isolated enhanced placental stem cells areisolated CD10⁺ CD33⁻, CD44⁺, CD45⁻, and CD117⁻ enhanced placental stemcells. In another embodiment, a cell population useful for the in themethods and compositions described herein is a population of cellscomprising, e.g., enriched for, isolated enhanced placental stem cells,wherein at least about 70%, at least about 80%, at least about 90%, atleast about 95% or at least about 99% of cells in said population ofcells are isolated CD10⁺ CD33⁻, CD44⁺, CD45⁻, and CD117⁻ enhancedplacental stem cells. In a specific embodiment, said isolated enhancedplacental stem cells or population of isolated enhanced placental stemcells is isolated away from placental cells that are not said cells. Inanother specific embodiment, said isolated enhanced placental stem cellsare non-maternal in origin. In another specific embodiment, at leastabout 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 90%, 85%, 90%, 95%, 98% or99% of said enhanced placental stem cells in said cell population arenon-maternal in origin. In another specific embodiment, said isolatedenhanced placental stem cells or population of isolated enhancedplacental stem cells is isolated away from placental cells that do notdisplay these markers.

In another embodiment, the isolated enhanced placental stem cells usefulin the methods and compositions described herein are isolated CD10⁺CD13⁻, CD33⁻, CD45⁻, and CD117⁻ enhanced placental stem cells. Inanother embodiment, a cell population useful in the methods andcompositions described herein is a population of cells comprising, e.g.,enriched for, isolated CD10⁺, CD13⁻, CD33⁻, CD45⁻, and CD117⁻ enhancedplacental stem cells, wherein at least about 70%, at least about 80%, atleast about 90%, at least about 95% or at least about 99% of cells insaid population are CD10⁺ CD13⁻, CD33⁻, CD45⁻, and CD117⁻ enhancedplacental stem cells. In a specific embodiment, said isolated enhancedplacental stem cells or population of isolated enhanced placental stemcells are isolated away from placental cells that are not said enhancedplacental stem cells. In another specific embodiment, said isolatedplacental cells are non-maternal in origin. In another specificembodiment, at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 90%,85%, 90%, 95%, 98% or 99% of said cells in said cell population arenon-maternal in origin. In another specific embodiment, said isolatedenhanced placental stem cells or population of isolated enhancedplacental stem cells is isolated away from placental cells that do notdisplay these characteristics.

In another embodiment, the isolated enhanced placental stem cells usefulin the methods and compositions described herein are HLA CD45⁻, CD34⁻,and CD133⁻, and are additionally CD10⁺, CD13⁺, CD38⁺, CD44⁺, CD90⁺,CD105⁺, CD200⁺ and/or HLA-G⁻, and/or negative for CD117. In anotherembodiment, a cell population useful in the methods described herein isa population of cells comprising isolated enhanced placental stem cells,wherein at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 98% or about 99% of the cells in saidpopulation are isolated enhanced placental stem cells that are HLAA,B,C⁻, CD45⁻, CD34⁻, CD133⁻, and that are additionally positive forCD10, CD13, CD38, CD44, CD90, CD105, CD200, and/or negative for CD117and/or HLA-G⁻. In a specific embodiment, said isolated enhancedplacental stem cells or population of isolated enhanced placental stemcells are isolated away from placental cells that are not said enhancedplacental stem cells. In another specific embodiment, said isolatedenhanced placental stem cells are non-maternal in origin. In anotherspecific embodiment, at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 90%, 85%, 90%, 95%, 98% or 99% of said enhanced placental stemcells in said cell population are non-maternal in origin. In anotherspecific embodiment, said isolated enhanced placental stem cells orpopulation of isolated enhanced placental stem cells are isolated awayfrom placental cells that do not display these characteristics.

In another embodiment, the isolated enhanced placental stem cells areisolated enhanced placental stem cells that are CD200⁺ and CD10⁺, asdetermined by antibody binding, and CD117⁻, as determined by bothantibody binding and RT-PCR. In another embodiment, the isolatedenhanced placental stem cells are isolated placental stem cells that areCD10⁺, CD29⁻, CD54⁺, CD200⁺, HLA-G⁻, MEW class I⁺ andβ-2-microglobulin⁺. In another embodiment, isolated enhanced placentalstem cells useful in the methods and compositions described herein areenhanced placental stem cells wherein the expression of at least onecellular marker is at least two-fold higher than in an equivalent numberof mesenchymal stem cells, e.g., bone marrow-derived mesenchymal stemcells. In another specific embodiment, said isolated enhanced placentalstem cells are non-maternal in origin. In another specific embodiment,at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 90%, 85%, 90%,95%, 98% or 99% of said cells in said cell population are non-maternalin origin.

In another embodiment, the isolated enhanced placental stem cells areisolated enhanced placental stem cells that are one or more of CD10⁺,CD29⁺, CD44⁺, CD45⁻, CD54/ICAM⁺, CD62E⁻, CD62L⁻, CD62P⁻, CD80⁻, CD86⁻,CD103⁻, CD104⁻, CD105⁺, CD106/VCAM⁺, CD144/VE-cadherin^(low),CD184/CXCR4⁻, β2-microglobulin^(low), MHC-II⁻, HLA-G^(low), and/orPDL1^(low). In a specific embodiment, the isolated enhanced placentalstem cells are at least CD29⁺ and CD54⁺. In another specific embodiment,the isolated enhanced placental stem cells are at least CD44⁺ andCD106⁺. In another specific embodiment, the isolated enhanced placentalstem cells are at least CD29⁺.

In another embodiment, a cell population useful in the methods andcompositions described herein comprises isolated enhanced placental stemcells, and at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% of thecells in said cell population are isolated enhanced placental stem cellsthat are one or more of CD10⁺, CD29⁺, CD44⁺, CD45⁻, CD54/ICAM⁺, CD62-E⁻,CD62-L⁻, CD62-P⁻, CD80⁻, CD86⁻, CD103⁻, CD104⁻, CD105⁺, CD106/VCAM⁺,CD144/VE-cadherin^(dim), CD184/CXCR4⁻, β2-microglobulin^(dim),HLA-I^(dim), HLA-II⁻, HLA-G^(dim), and/or PDL1^(dim) enhanced placentalstem cells. In another specific embodiment, at least 50%, 60%, 70%, 80%,90%, 95%, 98% or 99% of cells in said cell population are CD10⁺, CD29⁺,CD44⁺, CD45⁻, CD54/ICAM⁺, CD62-E⁻, CD62-L⁻, CD62-P⁻, CD80⁻, CD86⁻,CD103⁻, CD104⁻, CD105⁺, CD106/VCAM⁺, CD144/VE-cadherin^(dim),CD184/CXCR4⁻, β2-microglobulin^(dim), MHC-I^(dim), MHC-II⁻, HLA-G^(dim),and PDL1^(dim) enhanced placental stem cells. In certain embodiments,the enhanced placental stem cells express HLA-II markers when induced byinterferon gamma (IFN-γ).

In another embodiment, the isolated enhanced placental stem cells usefulin the methods and compositions described herein are isolated enhancedplacental stem cells that are one or more, or all, of CD10⁺, CD29⁺,CD34⁻, CD38⁻, CD44⁺, CD45⁻, CD54⁺, CD90⁺, SH2⁺, SH3⁺, SH4⁺, SSEA3⁻,SSEA4⁻, OCT-4⁺, and ABC-p⁺, where ABC-p is a placenta-specific ABCtransporter protein (also known as breast cancer resistance protein(BCRP) or as mitoxantrone resistance protein (MXR)), wherein saidisolated enhanced placental stem cells are derived from placental stemcells obtained by perfusion of a mammalian, e.g., human, placenta thathas been drained of cord blood and perfused to remove residual blood.

In another specific embodiment of any of the above embodiments,expression of the recited cellular marker(s) (e.g., cluster ofdifferentiation or immunogenic marker(s)) is determined by flowcytometry. In another specific embodiment, expression of the marker(s)is determined by RT-PCR.

Gene profiling confirms that isolated enhanced placental stem cells, andpopulations of isolated enhanced placental stem cells, aredistinguishable from other cells, e.g., mesenchymal stem cells, e.g.,bone marrow-derived mesenchymal stem cells. The isolated enhancedplacental stem cells described herein can be distinguished from, e.g.,bone marrow-derived mesenchymal stem cells on the basis of theexpression of one or more genes, the expression of which issignificantly higher in the isolated enhanced placental stem cells incomparison to bone marrow-derived mesenchymal stem cells. In particular,the isolated enhanced placental stem cells, useful in the methods oftreatment provided herein, can be distinguished from bone marrow-derivedmesenchymal stem cells on the basis of the expression of one or moregenes, the expression of which is significantly higher (that is, atleast twofold higher) in the isolated enhanced placental stem cells thanin an equivalent number of bone marrow-derived mesenchymal stem cells,wherein the one or more gene comprise ACTG2, ADARB1, AMIGO2, ARTS-1,B4GALT6, BCHE, C11orf9, CD200, COL4A1, COL4A2, CPA4, DMD, DSC3, DSG2,ELOVL2, F2RL1, FLJ10781, GATA6, GPR126, GPRC5B, ICAM1, IER3, IGFBP7,IL1A, IL6, IL18, KRT18, KRT8, LIPG, LRAP, MATN2, MEST, NFE2L3, NUAK1,PCDH7, PDLIM3, PKP2, RTN1, SERPINB9, ST3GAL6, ST6GALNAC5, SLC12A8,TCF21, TGFB2, VTN, ZC3H12A, or a combination of any of the foregoing,when the cells are grown under equivalent conditions. See, e.g., U.S.Patent Application Publication No. 2007/0275362, the disclosure of whichis incorporated herein by reference in its entirety. In certain specificembodiments, said expression of said one or more genes is determined,e.g., by RT-PCR or microarray analysis, e.g., using a U133-A microarray(Affymetrix).

In another specific embodiment, said isolated enhanced placental stemcells express said one or more genes when cultured for a number ofpopulation doublings, e.g., anywhere from about 3 to about 35 populationdoublings, in a medium comprising DMEM-LG (e.g., from Gibco); 2% fetalcalf serum (e.g., from Hyclone Labs.); 1× insulin-transferrin-selenium(ITS); 1× linoleic acid-bovine serum albumin (LA-BSA); 10⁻⁹Mdexamethasone (e.g., from Sigma); 10⁻⁴ M ascorbic acid 2-phosphate(e.g., from Sigma); epidermal growth factor 10 ng/mL (e.g., from R&DSystems); and platelet-derived growth factor (PDGF-BB) 10 ng/mL (e.g.,from R&D Systems). In another specific embodiment, the placentalcell-specific gene is CD200.

Specific sequences for these genes can be found in GenBank at accessionnos. NM_001615 (ACTG2), BC065545 (ADARB1), (NM_181847 (AMIGO2), AY358590(ARTS-1), BC074884 (B4GALT6), BC008396 (BCHE), BC020196 (C11orf9),BC031103 (CD200), NM_001845 (COL4A1), NM_001846 (COL4A2), BC052289(CPA4), BC094758 (DMD), AF293359 (DSC3), NM_001943 (DSG2), AF338241(ELOVL2), AY336105 (F2RL1), NM_018215 (FLJ10781), AY416799 (GATA6),BC075798 (GPR126), NM_016235 (GPRC5B), AF340038 (ICAM1), BC000844(IER3), BC066339 (IGFBP7), BC013142 (IL1A), BT019749 (IL6), BC007461(IL18), (BC072017) KRT18, BC075839 (KRT8), BC060825 (LIPG), BC065240(LRAP), BC010444 (MATN2), BC011908 (MEST), BC068455 (NFE2L3), NM_014840(NUAK1), AB006755 (PCDH7), NM_014476 (PDLIM3), BC126199 (PKP-2),BC090862 (RTN1), BC002538 (SERPINB9), BC023312 (ST3GAL6), BC001201(ST6GALNAC5), BC126160 or BC065328 (SLC12A8), BC025697 (TCF21), BC096235(TGFB2), BC005046 (VTN), and BC005001 (ZC3H12A) as of March 2008.

In certain specific embodiments, said isolated enhanced placental stemcells express each of ACTG2, ADARB1, AMIGO2, ARTS-1, B4GALT6, BCHE,C11orf9, CD200, COL4A1, COL4A2, CPA4, DMD, DSC3, DSG2, ELOVL2, F2RL1,F1110781, GATA6, GPR126, GPRC5B, ICAM1, IER3, IGFBP7, IL1A, IL6, IL18,KRT18, KRT8, LIPG, LRAP, MATN2, MEST, NFE2L3, NUAK1, PCDH7, PDLIM3,PKP2, RTN1, SERPINB9, ST3GAL6, ST6GALNAC5, SLC12A8, TCF21, TGFB2, VTN,and ZC3H12A at a detectably higher level than an equivalent number ofbone marrow-derived mesenchymal stem cells, when the cells are grownunder equivalent conditions.

In specific embodiments, the enhanced placental stem cells express CD200and ARTS1 (aminopeptidase regulator of type 1 tumor necrosis factor);ARTS-1 and LRAP (leukocyte-derived arginine aminopeptidase); IL6(interleukin-6) and TGFB2 (transforming growth factor, beta 2); IL6 andKRT18 (keratin 18); IER3 (immediate early response 3), MEST (mesodermspecific transcript homolog) and TGFB2; CD200 and IER3; CD200 and IL6;CD200 and KRT18; CD200 and LRAP; CD200 and MEST; CD200 and NFE2L3(nuclear factor (erythroid-derived 2)-like 3); or CD200 and TGFB2 at adetectably higher level than an equivalent number of bone marrow-derivedmesenchymal stem cells wherein said bone marrow-derived mesenchymal stemcells have undergone a number of passages in culture equivalent to thenumber of passages said isolated placental stem cells have undergone. Inother specific embodiments, the enhanced placental stem cells expressARTS-1, CD200, IL6 and LRAP; ARTS-1, IL6, TGFB2, IER3, KRT18 and MEST;CD200, IER3, IL6, KRT18, LRAP, MEST, NFE2L3, and TGFB2; ARTS-1, CD200,IER3, IL6, KRT18, LRAP, MEST, NFE2L3, and TGFB2; or IER3, MEST and TGFB2at a detectably higher level than an equivalent number of bonemarrow-derived mesenchymal stem cells, wherein said bone marrow-derivedmesenchymal stem cells have undergone a number of passages in cultureequivalent to the number of passages said isolated enhanced placentalstem cells have undergone.

Expression of the above-referenced genes can be assessed by standardtechniques. For example, probes based on the sequence of the gene(s) canbe individually selected and constructed by conventional techniques.Expression of the genes can be assessed, e.g., on a microarraycomprising probes to one or more of the genes, e.g., an AffymetrixGENECHIP® Human Genome U133A 2.0 array, or an Affymetrix GENECHIP® HumanGenome U133 Plus 2.0 (Santa Clara, Calif.). Expression of these genescan be assessed even if the sequence for a particular GenBank accessionnumber is amended because probes specific for the amended sequence canreadily be generated using well-known standard techniques.

The level of expression of these genes can be used to confirm theidentity of a population of isolated enhanced placental stem cells, toidentify a population of cells as comprising at least a plurality ofisolated enhanced placental stem cells, or the like. Populations ofisolated enhanced placental stem cells, the identity of which isconfirmed, can be clonal, e.g., populations of isolated enhancedplacental stem cells expanded from a single isolated enhanced placentalstem cell, or a mixed population of enhanced placental stem cells, e.g.,a population of cells comprising isolated enhanced placental stem cellsthat are expanded from multiple isolated enhanced placental stem cells,or a population of cells comprising isolated enhanced placental stemcells, as described herein, and at least one other type of cell.

The level of expression of these genes can be used to select populationsof isolated enhanced placental stem cells. For example, a population ofcells, e.g., clonally-expanded enhanced placental stem cells, may beselected if the expression of one or more of the genes listed above issignificantly higher in a sample from the population of cells than in anequivalent population of bone marrow-derived mesenchymal stem cells.Such selecting can be of a population from a plurality of isolatedenhanced placental stem cell populations, from a plurality of cellpopulations, the identity of which is not known, etc.

Isolated enhanced placental stem cells can be selected on the basis ofthe level of expression of one or more such genes as compared to thelevel of expression in said one or more genes in, e.g., a bonemarrow-derived mesenchymal stem cell control. In one embodiment, thelevel of expression of said one or more genes in a sample comprising anequivalent number of bone marrow-derived mesenchymal stem cells is usedas a control. In another embodiment, the control, for isolated enhancedplacental stem cells tested under certain conditions, is a numeric valuerepresenting the level of expression of said one or more genes in bonemarrow-derived mesenchymal stem cells under said conditions.

Similarly, the expression of survival-associated genes can be used toselect populations of isolated enhanced placental stem cells. Forexample, a population of cells, e.g., clonally-expanded enhancedplacental stem cells, may be selected if the expression of one or moresurvival-associated genes (e.g., one or more of the survival-associatedgenes described herein) is increased or decreased in a sample from thepopulation of cells relative an equivalent population of unmodifiedplacental stem cells.

The isolated enhanced placental stem cells described herein display theabove characteristics (e.g., combinations of cell surface markers and/orgene expression profiles) in primary culture, or during proliferation inmedium comprising, e.g., DMEM-LG (Gibco), 2% fetal calf serum (FCS)(Hyclone Laboratories), 1× insulin-transferrin-selenium (ITS), 1×linoleic-acid-bovine-serum-albumin (LA-BSA), 10⁻⁹M dexamethasone(Sigma), 10⁻⁴M ascorbic acid 2-phosphate (Sigma), epidermal growthfactor (EGF) 10 ng/ml (R&D Systems), platelet derived-growth factor(PDGF-BB) 10 ng/ml (R&D Systems), and 100 U penicillin/1000 Ustreptomycin.

In certain embodiments of any of the enhanced placental stem cellsdisclosed herein, the cells are human. In certain embodiments of any ofthe enhanced placental stem cells disclosed herein, the cellular markercharacteristics or gene expression characteristics are human markers orhuman genes.

In another specific embodiment of the isolated enhanced placental stemcells or populations of cells comprising the isolated enhanced placentalstem cells, said cells or population have been expanded, for example,passaged at least, about, or no more than, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times, or proliferated forat least, about, or no more than, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 or 40 populationdoublings. In another specific embodiment of said isolated enhancedplacental stem cells or populations of cells comprising the isolatedenhanced placental stem cells, said cells or population are primaryisolates. In another specific embodiment of the isolated enhancedplacental stem cells, or populations of cells comprising isolatedenhanced placental stem cells, that are disclosed herein, said isolatedenhanced placental stem cells are fetal in origin (that is, have thefetal genotype).

In certain embodiments, said isolated enhanced placental stem cells donot differentiate during culturing in growth medium, i.e., mediumformulated to promote proliferation, e.g., during proliferation ingrowth medium. In another specific embodiment, said isolated enhancedplacental stem cells do not require a feeder layer in order toproliferate. In another specific embodiment, said isolated enhancedplacental stem cells do not differentiate in culture in the absence of afeeder layer, solely because of the lack of a feeder cell layer.

In another embodiment, the isolated enhanced placental stem cells arepositive for aldehyde dehydrogenase (ALDH), as assessed by an aldehydedehydrogenase activity assay. Such assays are known in the art (see,e.g., Bostian and Betts, Biochem. J., 173, 787, (1978)). In a specificembodiment, said ALDH assay uses ALDEFLUOR® (Aldagen, Inc., Ashland,Oreg.) as a marker of aldehyde dehydrogenase activity. In a specificembodiment, between about 3% and about 25% of enhanced placental stemcells are positive for ALDH. In another embodiment, said isolatedenhanced placental stem cells show at least three-fold, or at leastfive-fold, higher ALDH activity than a population of bone marrow-derivedmesenchymal stem cells having about the same number of cells andcultured under the same conditions.

In certain embodiments of any of the populations of cells comprising theisolated enhanced placental stem cells described herein, the enhancedplacental stem cells in said populations of cells are substantially freeof cells having a maternal genotype; e.g., at least 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of the enhancedplacental stem cells in said population have a fetal genotype. Incertain other embodiments of any of the populations of cells comprisingthe isolated enhanced placental stem cells described herein, thepopulations of cells comprising said enhanced placental stem cells aresubstantially free of cells having a maternal genotype; e.g., at least40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%of the cells in said population have a fetal genotype.

In a specific embodiment of any of the above isolated enhanced placentalstem cells or cell populations comprising isolated enhanced placentalstem cells, the karyotype of the cells, e.g., all of the cells, or atleast about 95% or about 99% of the cells in said population, is normal.In another specific embodiment of any of the above enhanced placentalstem cells or populations or enhanced placental stem cells, the enhancedplacental stem cells are non-maternal in origin.

In a specific embodiment of any of the embodiments of placental cellsdisclosed herein, the placental cells are genetically stable, displayinga normal diploid chromosome count and a normal karyotype.

Isolated enhanced placental stem cells, or populations of isolatedenhanced placental stem cells, bearing any of the above combinations ofmarkers, can be combined in any ratio. Any two or more of the aboveisolated enhanced placental stem cells populations can be combined toform an isolated enhanced placental stem cell population. For example, apopulation of isolated enhanced placental stem cells can comprise afirst population of isolated enhanced placental stem cells defined byone of the marker combinations described above, and a second populationof isolated enhanced placental stem cells defined by another of themarker combinations described above, wherein said first and secondpopulations are combined in a ratio of about 1:99, 2:98, 3:97, 4:96,5:95, 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10,95:5, 96:4, 97:3, 98:2, or about 99:1. In like fashion, any three, four,five or more of the above-described isolated enhanced placental stemcells or isolated placental stem cell populations can be combined.

Isolated placental stem cells useful in methods for generating theenhanced placental stem cells described herein can be obtained, e.g., bydisruption of placental tissue, with or without enzymatic digestion orperfusion. For example, populations of isolated placental stem cells canbe produced according to a method comprising perfusing a mammalianplacenta that has been drained of cord blood and perfused to removeresidual blood; perfusing said placenta with a perfusion solution; andcollecting said perfusion solution, wherein said perfusion solutionafter perfusion comprises a population of placental cells that comprisesisolated placental stem cells; and isolating said placental stem cellsfrom said population of cells. In a specific embodiment, the perfusionsolution is passed through both the umbilical vein and umbilicalarteries and collected after it exudes from the placenta. In anotherspecific embodiment, the perfusion solution is passed through theumbilical vein and collected from the umbilical arteries, or passedthrough the umbilical arteries and collected from the umbilical vein.

In various embodiments, the isolated placental stem cells, useful inmethods for generating the enhanced placental stem cells describedherein contained within a population of cells obtained from perfusion ofa placenta, are at least 50%, 60%, 70%, 80%, 90%, 95%, 99% or at least99.5% of said population of placental stem cells. In another specificembodiment, the isolated placental stem cells collected by perfusioncomprise fetal and maternal cells. In another specific embodiment, theisolated placental stem cells collected by perfusion are at least 50%,60%, 70%, 80%, 90%, 95%, 99% or at least 99.5% fetal cells.

In another specific embodiment, provided herein is a compositioncomprising a population of the isolated placental stem cells useful inmethods for generating the enhanced placental stem cells describedherein, collected (isolated) by perfusion, wherein said compositioncomprises at least a portion of the perfusion solution used to isolatethe placental stem cells.

Populations of the isolated placental stem cells useful in methods forgenerating the enhanced placental stem cells described herein can beproduced by digesting placental tissue with a tissue-disrupting enzymeto obtain a population of placental cells comprising the placental stemcells, and isolating, or substantially isolating, a plurality of theplacental stem cells from the remainder of said placental cells. Thewhole, or any part of, the placenta can be digested to obtain theisolated placental stem cells described herein. In specific embodiments,for example, said placental tissue can be a whole placenta (e.g.,including an umbilical cord), an amniotic membrane, chorion, acombination of amnion and chorion, or a combination of any of theforegoing. In other specific embodiments, the tissue-disrupting enzymeis trypsin or collagenase. In various embodiments, the isolatedplacental stem cells, contained within a population of cells obtainedfrom digesting a placenta, are at least 50%, 60%, 70%, 80%, 90%, 95%,99% or at least 99.5% of said population of placental cells.

The populations of isolated enhanced placental stem cells describedabove, and populations of isolated enhanced placental stem cellsgenerally, can comprise about, at least, or no more than, 1×10⁵, 5×10⁵,1×10⁶, 5×10⁶, 1×10⁷, 5×10⁷, 1×10⁸, 5×10⁸, 1×10⁹, 5×10⁹, 1×10¹⁰, 5×10¹⁰,1×10¹¹ or more of the isolated enhanced placental stem cells.Populations of isolated enhanced placental stem cells useful in themethods and compositions described herein comprise at least 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% viable isolatedplacental stem cells, e.g., as determined by, e.g., trypan blueexclusion.

For any of the above placental stem cells, or populations of placentalstem cells, (e.g., unmodified placental stem cells useful in methods ofproducing the enhanced placental stem cells described herein, or theenhanced placental stem cells described herein, or compositions thereof)the cells or population of placental stem cells are, or can comprise,cells that have been passaged at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,12, 14, 16, 18, or 20 times, or more, or expanded for 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 or40 population doublings, or more.

In a specific embodiment of any of the above placental stem cells orplacental stem cells populations (e.g., unmodified placental stem cellsuseful in methods of producing the enhanced placental stem cellsdescribed herein, or the enhanced placental stem cells described herein,or compositions thereof), the karyotype of the cells, or at least about95% or about 99% of the cells in said population, is normal. In anotherspecific embodiment of any of the above placental stem cells orplacental stem cells populations (e.g., unmodified placental stem cellsuseful in methods of producing the enhanced placental stem cellsdescribed herein, or the enhanced placental stem cells described herein,or compositions thereof), the cells, or cells in the population ofcells, are non-maternal in origin.

Isolated placental stem cells, or populations of isolated placental stemcells, (e.g., unmodified placental stem cells useful in methods ofproducing the enhanced placental stem cells described herein, or theenhanced placental stem cells described herein, or compositions thereof)bearing any of the above combinations of markers, can be combined in anyratio. Any two or more of the above placental stem cells populations canbe isolated, or enriched, to form a placental stem cells population. Forexample, an population of isolated placental stem cells comprising afirst population of placental stem cells defined by one of the markercombinations described above can be combined with a second population ofplacental stem cells defined by another of the marker combinationsdescribed above, wherein said first and second populations are combinedin a ratio of about 1:99, 2:98, 3:97, 4:96, 5:95, 10:90, 20:80, 30:70,40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 96:4, 97:3, 98:2, orabout 99:1. In like fashion, any three, four, five or more of theabove-described placental stem cells or placental stem cells populationscan be combined.

In a specific embodiment of the above-mentioned placental stem cells(e.g., unmodified placental stem cells useful in methods of producingthe enhanced placental stem cells described herein, or the enhancedplacental stem cells described herein, or compositions thereof), theplacental stem cells constitutively secrete IL-6, IL-8 and monocytechemoattractant protein (MCP-1).

In certain embodiments, the enhanced placental stem cells useful in themethods provided herein, do not express CD34, as detected byimmunolocalization, after exposure to 1 to 100 ng/mL VEGF for 4 to 21days. In another specific embodiment, said enhanced placental stem cellsinduce endothelial cells to form sprouts or tube-like structures, e.g.,when cultured in the presence of an angiogenic factor such as vascularendothelial growth factor (VEGF), epithelial growth factor (EGF),platelet derived growth factor (PDGF) or basic fibroblast growth factor(bFGF), e.g., on a substrate such as MATRIGEL™.

In another aspect, the enhanced placental stem cells provided herein, ora population of cells, e.g., a population of enhanced placental stemcells, or a population of cells wherein at least about 50%, 60%, 70%,80%, 90%, 95% or 98% of cells in said population of cells are enhancedplacental stem cells, secrete one or more, or all, of VEGF, HGF, IL-8,MCP-3, FGF2, follistatin, G-CSF, EGF, ENA-78, GRO, IL-6, MCP-1, PDGF-BB,TIMP-2, uPAR, or galectin-1, e.g., into culture medium in which thecell, or cells, are grown. In another embodiment, the enhanced placentalstem cells express increased levels of CD202b, IL-8 and/or VEGF underhypoxic conditions (e.g., less than about 5% 02) compared to normoxicconditions (e.g., about 20% or about 21% 02).

In another embodiment, any of the enhanced placental stem cells orpopulations of cells comprising enhanced placental stem cells describedherein can cause the formation of sprouts or tube-like structures in apopulation of endothelial cells in contact with said enhanced placentalstem cells. In a specific embodiment, the enhanced placental stem cellsare co-cultured with human endothelial cells, which form sprouts ortube-like structures, or support the formation of endothelial cellsprouts, e.g., when cultured in the presence of extracellular matrixproteins such as collagen type I and IV, and/or angiogenic factors suchas vascular endothelial growth factor (VEGF), epithelial growth factor(EGF), platelet derived growth factor (PDGF) or basic fibroblast growthfactor (bFGF), e.g., in or on a substrate such as placental collagen orMATRIGEL™ for at least 4 days. In another embodiment, any of thepopulations of cells comprising enhanced placental stem cells describedherein secrete angiogenic factors such as vascular endothelial growthfactor (VEGF), hepatocyte growth factor (HGF), platelet derived growthfactor (PDGF), basic fibroblast growth factor (bFGF), or Interleukin-8(IL-8) and thereby can induce human endothelial cells to form sprouts ortube-like structures when cultured in the presence of extracellularmatrix proteins such as collagen type I and IV e.g., in or on asubstrate such as placental collagen or MATRIGEL™.

In another embodiment, any of the above populations of cells comprisingenhanced placental stem cells secretes angiogenic factors. In specificembodiments, the population of cells secretes vascular endothelialgrowth factor (VEGF), hepatocyte growth factor (HGF), platelet derivedgrowth factor (PDGF), basic fibroblast growth factor (bFGF), and/orinterleukin-8 (IL-8). In other specific embodiments, the population ofcells comprising enhanced placental stem cells secretes one or moreangiogenic factors and thereby induces human endothelial cells tomigrate in an in vitro wound healing assay. In other specificembodiments, the population of cells comprising enhanced placental stemcells induces maturation, differentiation or proliferation of humanendothelial cells, endothelial progenitors, myocytes or myoblasts.

In another embodiment, provided herein are enhanced placental stemcells, and populations of enhanced placental stem cells, wherein saidenhanced placental stem cells comprise any of the foregoingcharacteristics (e.g., are CD34⁻, CD10⁺, CD105⁺ and CD200⁺), and whereinat least one survival-associated gene is downregulated/inhibited in saidenhanced placental stem cells relative to the level of expression ofsaid survival-associated gene in an equivalent number of unmodifiedplacental stem cells (e.g., CD34⁻, CD10⁺, CD105⁺ and CD200⁺ unmodifiedplacental stem cells). In a specific embodiment, the at least onesurvival-associated gene is ADAMTS9, ABCF2, DNAJB4, MYB, RTN4, ANLN,BACE1, ABHD10, EGFR, NAA15, SEC24A MAP2K1, BCL2, ACTR1A, EIF4E, NAA25,SHOC2, CCNF, CAV2, ACVR2A, EPT1, NAPG, SLC12A2, CDC14A, CD276, ADSS,FGF2, NOB1, SLC16A3, CDC25A, CDC42, ALG3, FNDC3B, NOTCH2, SLC25A22,CHEK1, CDK6, ARHGDIA, GALNT7, SLC38A5, CUL2, COL3A1, ARL2, GPAM, PDCD4,SLC7A1, FGFR1, COL4A1, ATG9A, HACE1, PDCD6IP, SNX15, ITPR1, COL4A2,PLAG1, HARS, PHKB, SPTLC1, KIF23, CPEB3, C9ORF167/TOR4A, HARS2, PISD,SQSTM1, TRIM63, CXXC6/TET1, C9ORF89, HERC6, PLK1, SRPR, CSHL1, DIABLO,CACNA2D1, HMGA1, PNN, SRPRB, WEE1, DNMT3A, CAPRIN1, HSDL2, PNPLA6,TMEM43, MLLT1, DNMT3B, CCDC109A/MCU, IGF2R, PPIF, TNFSF9, MMS19, FGA,CCND1, IPO4, SIAH1, TOMM34, RECK, IMPDH1, CCND3, ITGA2, PPP2R5C, TPM3,RNASEL, INSIG1, CCNE1, KCNN4, PSAT1, TPPP3, WT1, KREMEN2, CCNT2, KPNA3,PTCD3, UBE2V1, YIF1B, LPL, CDC14B, LAMC1, PTGS2, UBE4A, ZNF622, MCL1,CDK5RAP1, LAMTOR3, PURA, UGDH, PIK3R1, CENPJ, LUZP1, RAB9B, UTP15,PPM1D, CHORDC1, LYPLA2, RAD51C, VEGFA, SPARC, CREBL2, PIAS1, RARS, orWNT3A. In a specific embodiment, 2, 3, 4, 5, 6, 7, 8, 9, 10, or morethan 10 of said survival-associated genes are downregulated/inhibited insaid enhanced placental stem cells relative to the level of expressionof said survival-associated gene (s) in an equivalent number ofunmodified placental stem cells (e.g., CD34⁻, CD10⁺, CD105⁺ and CD200⁺unmodified placental stem cells).

In another specific embodiment, provided herein is an isolated CD34⁻,CD10⁺, CD105⁺ and CD200⁺ enhanced placental stem cell, wherein saidenhanced placental stem cell expresses the survival-associated geneCCND1, CCND3, CCNE1, CCNF, CDK6, PPP2R5C, CDC25A, WEE1, CHEK1, MCL1,BCL2, PPMID, HMGA1, AKT3, VEGFA, MYB, or ITGA2 at a decreased level ascompared to the expression of the survival-associated gene in anunmodified CD34⁻, CD10⁺, CD105⁺ and CD200⁺ placental stem cell. Inanother specific embodiment, provided herein is an isolated CD34⁻,CD10⁺, CD105⁺ and CD200⁺ enhanced placental stem cell, wherein saidenhanced placental stem cell expresses one, two, three, or more of thefollowing placental stem cell survival-associated genes at a decreasedlevel as compared to the expression of the same survival-associated gene(s) in a corresponding unmodified placental stem cell: CCND1, CCND3,CCNE1, CCNF, CDK6, PPP2R5C, CDC25A, WEE1, CHEK1, MCL1, BCL2, PPMID,HMGA1, AKT3, VEGFA, MYB, or ITGA2. In another specific embodiment,provided herein is an isolated CD34⁻, CD10⁺, CD105⁺ and CD200⁺ enhancedplacental stem cell, wherein said enhanced placental stem cell (i)expresses one, two, three, or more of the following placental stem cellsurvival-associated genes at a decreased level as compared to theexpression of the same survival-associated gene (s) in a correspondingunmodified placental stem cell: CCND1, CCND3, CCNE1, CCNF, CDK6,PPP2R5C, CDC25A, WEE1, CHEK1, MCL1, BCL2, PPMID, HMGA1, AKT3, VEGFA,MYB, or ITGA2; and (ii) expresses at least one additionalsurvival-associated gene recited in Table 1 at an increased or adecreased level as compared to the expression of the samesurvival-associated gene (s) in a corresponding unmodified placentalstem cell. Further provided herein are populations of cells comprisingsuch enhanced placental stem cells and compositions comprising suchenhanced placental stem cells.

In another specific embodiment, provided herein is an isolated CD34⁻,CD10⁺, CD105⁺ and CD200⁺ enhanced placental stem cell, wherein saidenhanced placental stem cell expresses one, two, three, or more (i.e., acombination) of the following placental stem cell survival-associatedgenes at an increased level as compared to the expression of the samesurvival-associated gene(s) in a corresponding unmodified CD34⁻, CD10⁺,CD105⁺ and CD200⁺ placental stem cell: CCND1, CCND3, CCNE1, PPP2R5C,CDC25A, WEE1, MCL1, PPMID, HMGA1, AKT3, VEGFA, PPP2R5C, and/or ITGA2. Inanother specific embodiment, provided herein is an isolated CD34⁻,CD10⁺, CD105⁺ and CD200⁺ enhanced placental stem cell, wherein saidenhanced placental stem cell expresses one, two, three, or more of thefollowing placental stem cell survival-associated genes at an increasedlevel as compared to the expression of the same survival-associated gene(s) in a corresponding unmodified CD34⁻, CD10⁺, CD105⁺ and CD200⁺placental stem cell: CCND1, CCND3, CCNE1, PPP2R5C, CDC25A, WEE1, MCL1,PPMID, HMGA1, AKT3, VEGFA, PPP2R5C, and/or ITGA2. In another specificembodiment, provided herein is an isolated CD34⁻, CD10⁺, CD105⁺ andCD200⁺ enhanced placental stem cell, wherein said enhanced placentalstem cell (i) expresses one, two, three, or more of the followingplacental stem cell survival-associated genes at an increased level ascompared to the expression of the same survival-associated gene(s) in acorresponding unmodified CD34⁻, CD10⁺, CD105⁺ and CD200⁺ placental stemcell: CCND1, CCND3, CCNE1, PPP2R5C, CDC25A, WEE1, MCL1, PPMID, HMGA1,AKT3, VEGFA, PPP2R5C, and/or ITGA2; and (ii) expresses at least oneadditional survival-associated gene recited in Table 1 at an increasedor a decreased level as compared to the expression of the samesurvival-associated gene (s) in a corresponding unmodified placentalstem cell. Further provided herein are populations of cells comprisingsuch enhanced placental stem cells and compositions comprising suchenhanced placental stem cells.

5.3.3 Growth in Culture

The growth of placental cells, including the enhanced placental stemcells described herein, as for any mammalian cell, depends in part uponthe particular medium selected for growth. During culture, the placentalstem cells used in the methods of production of the enhanced placentalstem cells provided herein adhere to a substrate in culture, e.g. thesurface of a tissue culture container (e.g., tissue culture dishplastic, fibronectin-coated plastic, and the like) and form a monolayer.

In a specific embodiment, the enhanced placental stem cells describedherein demonstrate increased survival relative to correspondingunmodified placental stem cells when cultured under conditions thatcause cell death in vitro. In another specific embodiment, the enhancedplacental stem cells described herein demonstrate increased survivalrelative to corresponding unmodified placental stem cells when culturedunder conditions that cause cell death in vivo, e.g., when administeredto a subject.

In certain embodiments, when cultured under conditions that cause celldeath (either in vitro or in vivo), e.g., in the presence of serum(e.g., human or rat serum), complement, antibody(ies), other cells(e.g., cells of the immune system), and/or conditions that can lead toanoikis (e.g., low-attachment conditions) the enhanced placental stemcells described herein demonstrate at least a 1.5-fold, 2-fold,2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, or 10-fold increase in survival relative to anequivalent amount of corresponding unmodified placental stem cellscultured under the same conditions. In certain embodiments, whencultured under conditions that cause cell death (either in vitro or invivo), e.g., in the presence of serum (e.g., rat serum), complement,antibody(ies), other cells (e.g., cells of the immune system), and/orconditions that can lead to anoikis (e.g., low-attachment conditions)the enhanced placental stem cells described herein demonstrate a1.5-fold to 2.5-fold, a 2-fold to 3-fold, a 2.5-fold to 3.5-fold, a3-fold to 4-fold, a 3.5-fold to 4.5-fold, a 4-fold to 5-fold, a 5-foldto 6-fold, a 6-fold to 7-fold, a 7-fold to 8-fold, an 8-fold to 9-fold,or a 9-fold to 10-fold increase in survival relative to an equivalentamount of corresponding unmodified placental stem cells cultured underthe same conditions. Survival of the enhanced placental stem cells andunmodified placental stem cells can be assessed using methods known inthe art, e.g., trypan blue exclusion assay, fluorescein diacetate uptakeassay, propidium iodide uptake assay; thymidine uptake assay, and MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.

In certain embodiments, when cultured under conditions that cause celldeath (either in vitro or in vivo), e.g., in the presence of serum(e.g., rat serum), complement, antibody(ies), other cells (e.g., cellsof the immune system), and/or conditions that can lead to anoikis (e.g.,low-attachment conditions) the enhanced placental stem cells describedherein demonstrate (i) decreased caspase 3/7 activity, (ii) increasedmitochondrial membrane potential, and/or (iii) increased metabolicactivity as compared to corresponding unmodified placental stem cellscultured under the same condition(s). Caspase 3/7 activity,mitochondrial membrane potential, and metabolic activity can be assessedusing methods known in the art, e.g., as described in Sections 6.1.1.1.3and 6.1.1.2, below.

In certain embodiments, when cultured under conditions that cause celldeath (either in vitro or in vivo), e.g., in the presence of serum(e.g., rat serum), complement, antibody(ies), cells (e.g., cells of theimmune system), and/or conditions that lead to anoikis (e.g., lowattachment conditions) the enhanced placental stem cells describedherein exhibit (i) at least a 1.5-fold, 2-fold, 2.5-fold, 3-fold,3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or10-fold decrease in caspase 3/7 activity; (ii) at least a 1.5-fold,2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, or 10-fold increase in mitochondrial membranepotential; and/or (iii) at least a 1.5-fold, 2-fold, 2.5-fold, 3-fold,3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or10-fold increase in metabolic activity as compared to correspondingunmodified placental stem cells cultured under the same condition(s).

In certain embodiments, when cultured under conditions that cause celldeath (either in vitro or in vivo), e.g., in the presence of serum(e.g., rat serum), complement, antibody(ies), cells (e.g., cells of theimmune system), and/or conditions that can lead to anoikis (e.g.,low-attachment conditions) the enhanced placental stem cells describedherein exhibit (i) at least a 1.5-fold to 2.5-fold, a 2-fold to 3-fold,a 2.5-fold to 3.5-fold, a 3-fold to 4-fold, a 3.5-fold to 4.5-fold, a4-fold to 5-fold, a 5-fold to 6-fold, a 6-fold to 7-fold, a 7-fold to8-fold, an 8-fold to 9-fold, or a 9-fold to 10-fold decrease in caspase3/7 activity; (ii) at least a 1.5-fold to 2.5-fold, a 2-fold to 3-fold,a 2.5-fold to 3.5-fold, a 3-fold to 4-fold, a 3.5-fold to 4.5-fold, a4-fold to 5-fold, a 5-fold to 6-fold, a 6-fold to 7-fold, a 7-fold to8-fold, an 8-fold to 9-fold, or a 9-fold to 10-fold increase inmitochondrial membrane potential; and/or (iii) at least a 1.5-fold to2.5-fold, a 2-fold to 3-fold, a 2.5-fold to 3.5-fold, a 3-fold to4-fold, a 3.5-fold to 4.5-fold, a 4-fold to 5-fold, a 5-fold to 6-fold,a 6-fold to 7-fold, a 7-fold to 8-fold, an 8-fold to 9-fold, or a 9-foldto 10-fold increase in metabolic activity as compared to correspondingunmodified placental stem cells cultured under the same condition(s).

5.4 Methods of Obtaining Placental Stem Cells for Use in Methods ofGenerating Enhanced Placental Stem Cells

5.4.1 Stem Cell Collection Composition

Placental stem cells for use in the methods of generating enhancedplacental stem cells described herein can be collected and isolatedaccording to the methods provided herein. Generally, placental stemcells are obtained from a mammalian placenta using aphysiologically-acceptable solution, e.g., a stem cell collectioncomposition. A stem cell collection composition is described in detailin related U.S. Patent Application Publication No. 20070190042.

The stem cell collection composition can comprise anyphysiologically-acceptable solution suitable for the collection and/orculture of stem cells, for example, a saline solution (e.g.,phosphate-buffered saline, Kreb's solution, modified Kreb's solution,Eagle's solution, 0.9% NaCl. etc.), a culture medium (e.g., DMEM, HDMEM,etc.), and the like.

The stem cell collection composition can comprise one or more componentsthat tend to preserve placental stem cells, that is, prevent theplacental stem cells from dying, or delay the death of the placentalstem cells, reduce the number of placental stem cells in a population ofcells that die, or the like, from the time of collection to the time ofculturing. Such components can be, e.g., an apoptosis inhibitor (e.g., acaspase inhibitor or JNK inhibitor); a vasodilator (e.g., magnesiumsulfate, an antihypertensive drug, atrial natriuretic peptide (ANP),adrenocorticotropin, corticotropin-releasing hormone, sodiumnitroprusside, hydralazine, adenosine triphosphate, adenosine,indomethacin or magnesium sulfate, a phosphodiesterase inhibitor, etc.);a necrosis inhibitor (e.g., 2-(1H-Indol-3-yl)-3-pentylamino-maleimide,pyrrolidine dithiocarbamate, or clonazepam); a TNF-α inhibitor; and/oran oxygen-carrying perfluorocarbon (e.g., perfluorooctyl bromide,perfluorodecyl bromide, etc.).

The stem cell collection composition can comprise one or moretissue-degrading enzymes, e.g., a metalloprotease, a serine protease, aneutral protease, an RNase, or a DNase, or the like. Such enzymesinclude, but are not limited to, collagenases (e.g., collagenase I, II,III or IV, a collagenase from Clostridium histolyticum, etc.); dispase,thermolysin, elastase, trypsin, LIBERASE, hyaluronidase, and the like.

The stem cell collection composition can comprise a bacteriocidally orbacteriostatically effective amount of an antibiotic. In certainnon-limiting embodiments, the antibiotic is a macrolide (e.g.,tobramycin), a cephalosporin (e.g., cephalexin, cephradine, cefuroxime,cefprozil, cefaclor, cefixime or cefadroxil), a clarithromycin, anerythromycin, a penicillin (e.g., penicillin V) or a quinolone (e.g.,ofloxacin, ciprofloxacin or norfloxacin), a tetracycline, astreptomycin, etc. In a particular embodiment, the antibiotic is activeagainst Gram(+) and/or Gram(−) bacteria, e.g., Pseudomonas aeruginosa,Staphylococcus aureus, and the like.

The stem cell collection composition can also comprise one or more ofthe following compounds: adenosine (about 1 mM to about 50 mM);D-glucose (about 20 mM to about 100 mM); magnesium ions (about 1 mM toabout 50 mM); a macromolecule of molecular weight greater than 20,000daltons, in one embodiment, present in an amount sufficient to maintainendothelial integrity and cellular viability (e.g., a synthetic ornaturally occurring colloid, a polysaccharide such as dextran or apolyethylene glycol present at about 25 g/l to about 100 g/l, or about40 g/l to about 60 g/l); an antioxidant (e.g., butylated hydroxyanisole,butylated hydroxytoluene, glutathione, vitamin C or vitamin E present atabout 25 μM to about 100 μM); a reducing agent (e.g., N-acetylcysteinepresent at about 0.1 mM to about 5 mM); an agent that prevents calciumentry into cells (e.g., verapamil present at about 2 μM to about 25 μM);nitroglycerin (e.g., about 0.05 g/L to about 0.2 g/L); an anticoagulant,in one embodiment, present in an amount sufficient to help preventclotting of residual blood (e.g., heparin or hirudin present at aconcentration of about 1000 units/1 to about 100,000 units/1); or anamiloride containing compound (e.g., amiloride, ethyl isopropylamiloride, hexamethylene amiloride, dimethyl amiloride or isobutylamiloride present at about 1.0 μM to about 5 μM).

5.4.2 Collection and Handling of Placenta

Generally, a human placenta is recovered shortly after its expulsionafter birth. In a preferred embodiment, the placenta is recovered from apatient after informed consent and after a complete medical history ofthe patient is taken and is associated with the placenta. Preferably,the medical history continues after delivery. Such a medical history canbe used to coordinate subsequent use of the placenta or the stem cellsharvested therefrom. For example, human placental cells can be used, inlight of the medical history, for personalized medicine for the infantassociated with the placenta, or for parents, siblings or otherrelatives of the infant.

Prior to recovery of placental stem cells, the umbilical cord blood andplacental blood are removed. In certain embodiments, after delivery, thecord blood in the placenta is recovered. The placenta can be subjectedto a conventional cord blood recovery process. Typically a needle orcannula is used, with the aid of gravity, to exsanguinate the placenta(see, e.g., Anderson, U.S. Pat. No. 5,372,581; Hessel et al., U.S. Pat.No. 5,415,665). The needle or cannula is usually placed in the umbilicalvein and the placenta can be gently massaged to aid in draining cordblood from the placenta. Such cord blood recovery may be performedcommercially, e.g., LifeBank Inc., Cedar Knolls, N.J., ViaCord, CordBlood Registry and Cryocell. Preferably, the placenta is gravity drainedwithout further manipulation so as to minimize tissue disruption duringcord blood recovery.

Typically, a placenta is transported from the delivery or birthing roomto another location, e.g., a laboratory, for recovery of cord blood andcollection of stem cells by, e.g., perfusion or tissue dissociation. Theplacenta is preferably transported in a sterile, thermally insulatedtransport device (maintaining the temperature of the placenta between20-28° C.), for example, by placing the placenta, with clamped proximalumbilical cord, in a sterile zip-lock plastic bag, which is then placedin an insulated container. In another embodiment, the placenta istransported in a cord blood collection kit substantially as described inpending U.S. patent application Ser. No. 11/230,760, filed Sep. 19,2005. Preferably, the placenta is delivered to the laboratory four totwenty-four hours following delivery. In certain embodiments, theproximal umbilical cord is clamped, preferably within 4-5 cm(centimeter) of the insertion into the placental disc prior to cordblood recovery. In other embodiments, the proximal umbilical cord isclamped after cord blood recovery but prior to further processing of theplacenta.

The placenta, prior to placental stem cell collection, can be storedunder sterile conditions and at either room temperature or at atemperature of 5 to 25° C. (centigrade). The placenta may be stored fora period of longer than forty eight hours, and preferably for a periodof four to twenty-four hours prior to perfusing the placenta to removeany residual cord blood. The placenta is preferably stored in ananticoagulant solution at a temperature of 5 to 25° C. (centigrade).Suitable anticoagulant solutions are well known in the art. For example,a solution of heparin or warfarin sodium can be used. In a preferredembodiment, the anticoagulant solution comprises a solution of heparin(e.g., 1% w/w in 1:1000 solution). The exsanguinated placenta ispreferably stored for no more than 36 hours before placental cells arecollected.

The mammalian placenta or a part thereof, once collected and preparedgenerally as above, can be treated in any art-known manner, e.g., can beperfused or disrupted, e.g., digested with one or more tissue-disruptingenzymes, to obtain stem cells.

5.4.3 Physical Disruption and Enzymatic Digestion of Placental Tissue

In one embodiment, placental stem cells are collected from a mammalianplacenta by physical disruption, e.g., enzymatic digestion, of theorgan, e.g., using the stem cell collection composition described above.For example, the placenta, or a portion thereof, may be, e.g., crushed,sheared, minced, diced, chopped, macerated or the like, while in contactwith, e.g., a buffer, medium or a stem cell collection composition, andthe tissue subsequently digested with one or more enzymes. The placenta,or a portion thereof, may also be physically disrupted and digested withone or more enzymes, and the resulting material then immersed in, ormixed into, a buffer, medium or a stem cell collection composition. Anymethod of physical disruption can be used, provided that the method ofdisruption leaves a plurality, more preferably a majority, and morepreferably at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the cells insaid organ viable, as determined by, e.g., trypan blue exclusion.

Typically, placental cells can be obtained by disruption of a smallblock of placental tissue, e.g., a block of placental tissue that isabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 200, 300, 400, 500, 600, 700, 800, 900 or about 1000 cubicmillimeters in volume.

Enzymatic digestion can be performed using single enzymes orcombinations of enzymes. In one embodiment, enzymatic digestion ofplacental tissue uses a combination of a matrix metalloprotease, aneutral protease, and a mucolytic enzyme for digestion of hyaluronicacid, such as a combination of collagenase, dispase, and hyaluronidaseor a combination of LIBERASE (Boehringer Mannheim Corp., Indianapolis,Ind.) and hyaluronidase. Other enzymes that can be used to disruptplacenta tissue include papain, deoxyribonucleases, serine proteases,such as trypsin, chymotrypsin, or elastase. Serine proteases may beinhibited by alpha 2 microglobulin in serum and therefore the mediumused for digestion is usually serum-free. EDTA and DNase are commonlyused in enzyme digestion procedures to increase the efficiency of cellrecovery. The digestate is preferably diluted so as to avoid trappingstem cells within the viscous digest.

Typical concentrations for tissue digestion enzymes include, e.g.,50-200 U/mL for collagenase I and collagenase IV, 1-10 U/mL for dispase,and 10-100 U/mL for elastase. Proteases can be used in combination, thatis, two or more proteases in the same digestion reaction, or can be usedsequentially in order to liberate placental cells. For example, in oneembodiment, a placenta, or part thereof, is digested first with anappropriate amount of collagenase I at 2 mg/ml for 30 minutes, followedby digestion with trypsin, 0.25%, for 10 minutes, at 37° C. Serineproteases are preferably used consecutively following use of otherenzymes.

In another embodiment, the tissue can further be disrupted by theaddition of a chelator, e.g., ethylene glycol bis(2-aminoethylether)-N,N,N′N′-tetraacetic acid (EGTA) or ethylenediaminetetraaceticacid (EDTA) to the stem cell collection composition comprising the stemcells, or to a solution in which the tissue is disrupted and/or digestedprior to isolation of the placental stem cells with the stem cellcollection composition.

It will be appreciated that where an entire placenta, or portion of aplacenta comprising both fetal and maternal cells (for example, wherethe portion of the placenta comprises the chorion or cotyledons) isdigested to obtain placental stem cells, the placental cells collectedwill comprise a mix of placental cells derived from both fetal andmaternal sources. Where a portion of the placenta that comprises no, ora negligible number of, maternal cells (for example, amnion) is used toobtain placental stem cells, the placental stem cells collected willcomprise almost exclusively fetal placental stem cells.

5.4.4 Placental Perfusion

Placental stem cells can also be obtained by perfusion of the mammalianplacenta. Methods of perfusing mammalian placenta to obtain stem cellsare disclosed, e.g., in U.S. Pat. No. 7,045,148.

Placental stem cells can be collected by perfusion, e.g., through theplacental vasculature, using, e.g., a stem cell collection compositionas a perfusion solution. In one embodiment, a mammalian placenta isperfused by passage of perfusion solution through either or both of theumbilical artery and umbilical vein. The flow of perfusion solutionthrough the placenta may be accomplished using, e.g., gravity flow intothe placenta. Preferably, the perfusion solution is forced through theplacenta using a pump, e.g., a peristaltic pump. The umbilical vein canbe, e.g., cannulated with a cannula, e.g., a TEFLON® or plastic cannula,that is connected to a sterile connection apparatus, such as steriletubing. The sterile connection apparatus is connected to a perfusionmanifold.

In preparation for perfusion, the placenta is preferably oriented (e.g.,suspended) in such a manner that the umbilical artery and umbilical veinare located at the highest point of the placenta. The placenta can beperfused by passage of a perfusion fluid, e.g., the stem cell collectioncomposition provided herein, through the placental vasculature, orthrough the placental vasculature and surrounding tissue. In oneembodiment, the umbilical artery and the umbilical vein are connectedsimultaneously to a pipette that is connected via a flexible connectorto a reservoir of the perfusion solution. The perfusion solution ispassed into the umbilical vein and artery. The perfusion solution exudesfrom and/or passes through the walls of the blood vessels into thesurrounding tissues of the placenta, and is collected in a suitable openvessel from the surface of the placenta that was attached to the uterusof the mother during gestation. The perfusion solution may also beintroduced through the umbilical cord opening and allowed to flow orpercolate out of openings in the wall of the placenta which interfacedwith the maternal uterine wall. In another embodiment, the perfusionsolution is passed through the umbilical veins and collected from theumbilical artery, or is passed through the umbilical artery andcollected from the umbilical veins.

In one embodiment, the proximal umbilical cord is clamped duringperfusion, and more preferably, is clamped within 4-5 cm (centimeter) ofthe cord's insertion into the placental disc.

The first collection of perfusion fluid from a mammalian placenta duringthe exsanguination process is generally colored with residual red bloodcells of the cord blood and/or placental blood; this portion of theperfusion can be discarded. The perfusion fluid becomes more colorlessas perfusion proceeds and the residual cord blood cells are washed outof the placenta.

The volume of perfusion liquid used to collect placental stem cells mayvary depending upon the number of placental stem cells to be collected,the size of the placenta, the number of collections to be made from asingle placenta, etc. In various embodiments, the volume of perfusionliquid may be from 50 mL to 5000 mL, 50 mL to 4000 mL, 50 mL to 3000 mL,100 mL to 2000 mL, 250 mL to 2000 mL, 500 mL to 2000 mL, or 750 mL to2000 mL. Typically, the placenta is perfused with 700-800 mL ofperfusion liquid following exsanguination.

The placenta can be perfused a plurality of times over the course ofseveral hours or several days. Where the placenta is to be perfused aplurality of times, it may be maintained or cultured under asepticconditions in a container or other suitable vessel, and perfused withthe stem cell collection composition, or a standard perfusion solution(e.g., a normal saline solution such as phosphate buffered saline(“PBS”)) with or without an anticoagulant (e.g., heparin, warfarinsodium, coumarin, bishydroxycoumarin), and/or with or without anantimicrobial agent (e.g., β-mercaptoethanol (0.1 mM); antibiotics suchas streptomycin (e.g., at 40-100 μg/ml), penicillin (e.g., at 40 U/ml),amphotericin B (e.g., at 0.5 μg/ml). In one embodiment, an isolatedplacenta is maintained or cultured for a period of time withoutcollecting the perfusate, such that the placenta is maintained orcultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, or 24 hours, or 2 or 3 or more days beforeperfusion and collection of perfusate. The perfused placenta can bemaintained for one or more additional time(s), e.g., 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 ormore hours, and perfused a second time with, e.g., 700-800 mL perfusionfluid. The placenta can be perfused 1, 2, 3, 4, 5 or more times, forexample, once every 1, 2, 3, 4, 5 or 6 hours. In a preferred embodiment,perfusion of the placenta and collection of perfusion solution, e.g.,stem cell collection composition, is repeated until the number ofrecovered nucleated cells falls below 100 cells/ml. The perfusates atdifferent time points can be further processed individually to recovertime-dependent populations of placental stem cells. Perfusates fromdifferent time points can also be pooled.

Without wishing to be bound by any theory, after exsanguination and asufficient time of perfusion of the placenta, placental stem cells arebelieved to migrate into the exsanguinated and perfused microcirculationof the placenta where they are collectable, preferably by washing into acollecting vessel by perfusion. Perfusing the isolated placenta not onlyserves to remove residual cord blood but also provide the placenta withthe appropriate nutrients, including oxygen. The placenta may becultivated and perfused with a similar solution which was used to removethe residual cord blood cells, preferably, without the addition ofanticoagulant agents.

Stem cells can be isolated from placenta by perfusion with a solutioncomprising one or more proteases or other tissue-disruptive enzymes. Ina specific embodiment, a placenta or portion thereof is brought to25-37° C., and is incubated with one or more tissue-disruptive enzymesin 200 mL of a culture medium for 30 minutes. Cells from the perfusateare collected, brought to 4° C., and washed with a cold inhibitor mixcomprising 5 mM EDTA, 2 mM dithiothreitol and 2 mM beta-mercaptoethanol.The placental stem cells are washed after several minutes with a cold(e.g., 4° C.) stem cell collection composition described elsewhereherein.

Perfusion using the pan method, that is, whereby perfusate is collectedafter it has exuded from the maternal side of the placenta, results in amix of fetal and maternal cells. As a result, the cells collected bythis method comprise a mixed population of placental stem cells of bothfetal and maternal origin. In contrast, perfusion solely through theplacental vasculature, whereby perfusion fluid is passed through one ortwo placental vessels and is collected solely through the remainingvessel(s), results in the collection of a population of placental stemcells almost exclusively of fetal origin.

5.4.5 Isolation, Sorting, and Characterization of Placental Cells

Stem cells from mammalian placenta, whether obtained by perfusion orenyzmatic digestion, can initially be purified from (i.e., be isolatedfrom) other cells by Ficoll gradient centrifugation. Such centrifugationcan follow any standard protocol for centrifugation speed, etc. In oneembodiment, for example, cells collected from the placenta are recoveredfrom perfusate by centrifugation at 5000×g for 15 minutes at roomtemperature, which separates cells from, e.g., contaminating debris andplatelets. In another embodiment, placental perfusate is concentrated toabout 200 ml, gently layered over Ficoll, and centrifuged at about1100×g for 20 minutes at 22° C., and the low-density interface layer ofcells is collected for further processing.

Cell pellets can be resuspended in fresh stem cell collectioncomposition, or a medium suitable for stem cell maintenance, e.g., IMDMserum-free medium containing 2 U/ml heparin and 2 mM EDTA (GibcoBRL,NY). The total mononuclear cell fraction can be isolated, e.g., usingLymphoprep (Nycomed Pharma, Oslo, Norway) according to themanufacturer's recommended procedure.

As used herein, “isolating” placental stem cells means removing at least20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the cells withwhich the placental stem cells are normally associated in the intactmammalian placenta.

Placental stem cells obtained by perfusion or digestion can, forexample, be further, or initially, isolated by differentialtrypsinization using, e.g., a solution of 0.05% trypsin with 0.2% EDTA(Sigma, St. Louis Mo.). Differential trypsinization is possible becauseplacental stem cells typically detach from plastic surfaces within aboutfive minutes whereas other adherent populations typically require morethan 20-30 minutes incubation. The detached placental stem cells can beharvested following trypsinization and trypsin neutralization, using,e.g., Trypsin Neutralizing Solution (TNS, Cambrex).

In one embodiment of isolation of placental stem cells, aliquots of, forexample, about 5-10×10⁶ placental cells are placed in each of severalT-75 flasks, preferably fibronectin-coated T75 flasks. In such anembodiment, the cells can be cultured with commercially availableMesenchymal Stem Cell Growth Medium (MSCGM) (Cambrex), and placed in atissue culture incubator (37° C., 5% CO₂). After 10 to 15 days,non-adherent cells are removed from the flasks by washing with PBS. ThePBS is then replaced by MSCGM. Flasks are preferably examined daily forthe presence of various adherent cell types and in particular, foridentification and expansion of clusters of fibroblastoid cells.

The number and type of cells collected from a mammalian placenta can bemonitored, for example, by measuring changes in morphology and cellsurface markers using standard cell detection techniques such as flowcytometry, cell sorting, immunocytochemistry (e.g., staining with tissuespecific or cell-marker specific antibodies) fluorescence activated cellsorting (FACS), magnetic activated cell sorting (MACS), by examinationof the morphology of cells using light or confocal microscopy, and/or bymeasuring changes in gene expression using techniques well known in theart, such as PCR and gene expression profiling. These techniques can beused, too, to identify cells that are positive for one or moreparticular markers. For example, using antibodies to CD34, one candetermine, using the techniques above, whether a cell comprises adetectable amount of CD34 as compared to, for example, an isotypecontrol; if so, the cell is CD34⁺. Likewise, if a cell produces enoughOCT-4 RNA to be detectable by RT-PCR, or significantly more OCT-4 RNAthan a terminally-differentiated cell, the cell is OCT-4⁺. Antibodies tocell surface markers (e.g., CD markers such as CD34) and the sequence ofstem cell-specific genes, such as OCT-4, are well-known in the art.

Placental cells, particularly cells that have been isolated by Ficollseparation, differential adherence, or a combination of both, may besorted, e.g., further isolated, using a fluorescence activated cellsorter (FACS). Fluorescence activated cell sorting (FACS) is awell-known method for separating particles, including cells, based onthe fluorescent properties of the particles (Kamarch, 1987, MethodsEnzymol, 151:150-165). Laser excitation of fluorescent moieties in theindividual particles results in a small electrical charge allowingelectromagnetic separation of positive and negative particles from amixture. In one embodiment, cell surface marker-specific antibodies orligands are labeled with distinct fluorescent labels. Cells areprocessed through the cell sorter, allowing separation of cells based ontheir ability to bind to the antibodies used. FACS sorted particles maybe directly deposited into individual wells of 96-well or 384-wellplates to facilitate separation and cloning.

In one sorting scheme, placental stem cells can be sorted on the basisof expression of the markers CD34, CD38, CD44, CD45, CD73, CD105, OCT-4and/or HLA-G, or any of the other markers listed elsewhere herein. Thiscan be accomplished in connection with procedures to select stem cellson the basis of their adherence properties in culture. For example,adherence selection of placental stem cells can be accomplished beforeor after sorting on the basis of marker expression. In one embodiment,for example, placental stem cells can be sorted first on the basis oftheir expression of CD34; CD34⁻ cells are retained, and cells that areCD200⁺ or HLA-G⁻, are separated from all other CD34⁻ cells. In anotherembodiment, placental stem cells can be sorted based on their expressionof CD200 and/or HLA-G, or lack thereof; for example, cells displayingeither of these markers can be isolated for further use. Cells thatexpress, e.g., CD200 and/or HLA-G can, in a specific embodiment, befurther sorted based on their expression of CD73 and/or CD105, orepitopes recognized by antibodies SH2, SH3 or SH4, or lack of expressionof CD34, CD38 or CD45. For example, in one embodiment, placental stemcells are sorted by expression, or lack thereof, of CD200, HLA-G, CD73,CD105, CD34, CD38 and CD45, and placental stem cells that are CD200⁺,HLA-G⁻, CD73⁺, CD105⁺, CD34⁻, CD38⁻ and CD45⁻ are isolated from otherplacental cells for further use.

In another embodiment, magnetic beads can be used to separate cells,e.g., separate placental stem cells from other placental cells. Thecells may be sorted using a magnetic activated cell sorting (MACS)technique, a method for separating particles based on their ability tobind magnetic beads (0.5-100 μm diameter). A variety of usefulmodifications can be performed on the magnetic microspheres, includingcovalent addition of antibody that specifically recognizes a particularcell surface molecule or hapten. The beads are then mixed with the cellsto allow binding. Cells are then passed through a magnetic field toseparate out cells having the specific cell surface marker. In oneembodiment, these cells can then isolated and re-mixed with magneticbeads coupled to an antibody against additional cell surface markers.The cells are again passed through a magnetic field, isolating cellsthat bound both the antibodies. Such cells can then be diluted intoseparate dishes, such as microtiter dishes for clonal isolation.

Placental stem cells can also be characterized and/or sorted based oncell morphology and growth characteristics. For example, placental stemcells can be characterized as having, and/or selected on the basis of,e.g., a fibroblastoid appearance in culture. Placental stem cells canalso be characterized as having, and/or be selected, on the basis oftheir ability to form embryoid-like bodies. In one embodiment, forexample, placental cells that are fibroblastoid in shape, express CD73and CD105, and produce one or more embryoid-like bodies in culture canbe isolated from other placental cells. In another embodiment, OCT-4⁺placental cells that produce one or more embryoid-like bodies in cultureare isolated from other placental cells.

In another embodiment, placental stem cells can be identified andcharacterized by a colony forming unit assay. Colony forming unit assaysare commonly known in the art, such as Mesen Cult™ medium (Stem CellTechnologies, Inc., Vancouver British Columbia).

Placental stem cells can be assessed for viability, proliferationpotential, and longevity using standard techniques known in the art,such as trypan blue exclusion assay, fluorescein diacetate uptake assay,propidium iodide uptake assay (to assess viability); and thymidineuptake assay, MTT cell proliferation assay (to assess proliferation).Longevity may be determined by methods well known in the art, such as bydetermining the maximum number of population doubling in an extendedculture.

Placental stem cells can also be separated from other placental cellsusing other techniques known in the art, e.g., selective growth ofdesired cells (positive selection), selective destruction of unwantedcells (negative selection); separation based upon differential cellagglutinability in the mixed population as, for example, with soybeanagglutinin; freeze-thaw procedures; filtration; conventional and zonalcentrifugation; centrifugal elutriation (counter-streamingcentrifugation); unit gravity separation; countercurrent distribution;electrophoresis; and the like.

5.5 Culture of Placental Stem Cells

5.5.1 Culture Media

Placental stem cells, including the enhanced placental stem cellsdescribed herein, can be cultured in any medium, and under anyconditions, recognized in the art as acceptable for the culture of stemcells. In certain embodiments, the culture medium comprises serum. Incertain embodiments, placental stem cells, including the enhancedplacental stem cells described herein, can be cultured in, for example,DMEM-LG (Dulbecco's Modified Essential Medium, low glucose)/MCDB 201(chick fibroblast basal medium) containing ITS(insulin-transferrin-selenium), LA+BSA (linoleic acid-bovine serumalbumin), dextrose, L-ascorbic acid, PDGF, EGF, IGF-1, andpenicillin/streptomycin; DMEM-HG (high glucose) comprising 10% fetalbovine serum (FBS); DMEM-HG comprising 15% FBS; IMDM (Iscove's modifiedDulbecco's medium) comprising 10% FBS, 10% horse serum, andhydrocortisone; M199 comprising 10% FBS, EGF, and heparin; α-MEM(minimal essential medium) comprising 10% FBS, GlutaMAX™ and gentamicin;DMEM comprising 10% FBS, GlutaMAX™ and gentamicin, etc. A preferredmedium is DMEM-LG/MCDB-201 comprising 2% FBS, ITS, LA+BSA, dextrose,L-ascorbic acid, PDGF, EGF, and penicillin/streptomycin.

Other media in that can be used to culture placental stem cells,including the enhanced placental stem cells described herein, includeDMEM (high or low glucose), Eagle's basal medium, Ham's F10 medium(F10), Ham's F-12 medium (F12), Iscove's modified Dulbecco's medium,Mesenchymal Stem Cell Growth Medium (MSCGM), Liebovitz's L-15 medium,MCDB, DMIEM/F12, RPMI 1640, advanced DMEM (Gibco), DMEM/MCDB201 (Sigma),and CELL-GRO FREE.

The culture medium can be supplemented with one or more componentsincluding, for example, serum (e.g., fetal bovine serum (FBS),preferably about 2-15% (v/v); equine (horse) serum (ES); human serum(HS)); beta-mercaptoethanol (BME), preferably about 0.001% (v/v); one ormore growth factors, for example, platelet-derived growth factor (PDGF),epidermal growth factor (EGF), basic fibroblast growth factor (bFGF),insulin-like growth factor-1 (IGF-1), leukemia inhibitory factor (LIF),vascular endothelial growth factor (VEGF), and erythropoietin (EPO);amino acids, including L-valine; and one or more antibiotic and/orantimycotic agents to control microbial contamination, such as, forexample, penicillin G, streptomycin sulfate, amphotericin B, gentamicin,and nystatin, either alone or in combination.

5.5.2 Expansion and Proliferation of Placental Stem Cells

Once placental stem cells, including the enhanced placental stem cellsdescribed herein, are isolated, the stem cells or population of stemcells can be proliferated and expanded in vitro. For example, onceenhanced placental stem cells are produced, such cells can also beproliferated and expanded in vitro. Placental stem cells, including theenhanced placental stem cells described herein, can be cultured intissue culture containers, e.g., dishes, flasks, multiwell plates, orthe like, for a sufficient time for the placental stem cells toproliferate to 70-90% confluence, that is, until the placental stemcells and their progeny occupy 70-90% of the culturing surface area ofthe tissue culture container.

Placental stem cells, including the enhanced placental stem cellsdescribed herein, can be seeded in culture vessels at a density thatallows cell growth. For example, the placental stem cells may be seededat low density (e.g., about 1,000 to about 5,000 cells/cm²) to highdensity (e.g., about 50,000 or more cells/cm²). In a preferredembodiment, the placental stem cells are cultured at about 0 to about 5percent by volume CO₂ in air. In some preferred embodiments, theplacental stem cells are cultured at about 2 to about 25 percent O₂ inair, preferably about 5 to about 20 percent O₂ in air. The placentalstem cells preferably are cultured at about 25° C. to about 40° C.,preferably 37° C. The placental stem cells are preferably cultured in anincubator. The culture medium can be static or agitated, for example,using a bioreactor. Placental stem cells can be grown under lowoxidative stress (e.g., with addition of glutathione, ascorbic acid,catalase, tocopherol, N-acetylcysteine, or the like).

Once 70%-90% confluence is obtained, the placental stem cells, includingthe enhanced placental stem cells described herein, may be passaged. Forexample, the cells can be enzymatically treated, e.g., trypsinized,using techniques well-known in the art, to separate them from the tissueculture surface. After removing the placental stem cells by pipettingand counting the cells, about 20,000-100,000 stem cells, preferablyabout 50,000 placental stem cells, are passaged to a new culturecontainer containing fresh culture medium. Typically, the new medium isthe same type of medium from which the stem cells were removed. Providedherein are populations of placental stem cells, including the enhancedplacental stem cells described herein, that have been passaged at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 times, or more, andcombinations of the same.

5.6 Preservation of Enhanced Placental Cells

Enhanced placental stem cells can be preserved, that is, placed underconditions that allow for long-term storage, or conditions that inhibitcell death by, e.g., apoptosis or necrosis.

Enhanced placental stem cells can be preserved using, e.g., acomposition comprising an apoptosis inhibitor, necrosis inhibitor and/oran oxygen-carrying perfluorocarbon, as described in related U.S. PatentApplication Publication No. 2007/0190042.

In one embodiment, provided herein is a method of preserving enhancedplacental stem cells comprising contacting said enhanced placental stemcells with a stem cell collection composition comprising an inhibitor ofapoptosis and an oxygen-carrying perfluorocarbon, wherein said inhibitorof apoptosis is present in an amount and for a time sufficient to reduceor prevent apoptosis in the population of enhanced placental stem cells,as compared to a population of enhanced placental stem cells notcontacted with the inhibitor of apoptosis. In a specific embodiment,said inhibitor of apoptosis is a caspase inhibitor. In another specificembodiment, said inhibitor of apoptosis is a JNK inhibitor. In a morespecific embodiment, said JNK inhibitor does not modulatedifferentiation or proliferation of said enhanced placental stem cells.In another embodiment, said stem cell collection composition comprisessaid inhibitor of apoptosis and said oxygen-carrying perfluorocarbon inseparate phases. In another embodiment, said stem cell collectioncomposition comprises said inhibitor of apoptosis and saidoxygen-carrying perfluorocarbon in an emulsion. In another embodiment,the stem cell collection composition additionally comprises anemulsifier, e.g., lecithin. In another embodiment, said apoptosisinhibitor and said perfluorocarbon are between about 0° C. and about 25°C. at the time of contacting the stem cells. In another more specificembodiment, said apoptosis inhibitor and said perfluorocarbon arebetween about 2° C. and 10° C., or between about 2° C. and about 5° C.,at the time of contacting the stem cells. In another more specificembodiment, said contacting is performed during transport of saidenhanced placental stem cells. In another more specific embodiment, saidcontacting is performed during freezing and thawing of said populationof enhanced placental stem cells.

In another embodiment, enhanced placental stem cells can be preserved bya method comprising contacting said enhanced placental stem cells withan inhibitor of apoptosis and an organ-preserving compound, wherein saidinhibitor of apoptosis is present in an amount and for a time sufficientto reduce or prevent apoptosis of the enhanced placental stem cells, ascompared to enhanced placental stem cells not contacted with theinhibitor of apoptosis. In a specific embodiment, the organ-preservingcompound is UW solution (described in U.S. Pat. No. 4,798,824; alsoknown as ViaSpan; see also Southard et al., Transplantation49(2):251-257 (1990)) or a solution described in Stern et al., U.S. Pat.No. 5,552,267. In another embodiment, said organ-preserving compound ishydroxyethyl starch, lactobionic acid, raffinose, or a combinationthereof.

In another embodiment, placental stem cells, to be used to produceenhanced placental stem cells, are contacted with a stem cell collectioncomposition comprising an apoptosis inhibitor and oxygen-carryingperfluorocarbon, organ-preserving compound, or combination thereof,during perfusion. In another embodiment, said placental stem cells, tobe used to produce enhanced placental stem cells, are contacted during aprocess of tissue disruption, e.g., enzymatic digestion. In anotherembodiment, placental cells, to be used to produce enhanced placentalstem cells, are contacted with said stem cell collection compound aftercollection by perfusion, or after collection by tissue disruption, e.g.,enzymatic digestion.

Typically, during placental stem cell collection, enrichment andisolation, it is preferable to minimize or eliminate cell stress due tohypoxia and mechanical stress. In another embodiment of the method,therefore, placental stem cells, to be used to produce enhancedplacental stem cells, are exposed to a hypoxic condition duringcollection, enrichment or isolation for less than six hours during saidpreservation, wherein a hypoxic condition is a concentration of oxygenthat is less than normal blood oxygen concentration. In a more specificembodiment, said placental stem cells are exposed to said hypoxiccondition for less than two hours during said preservation. In anothermore specific embodiment, said placental stem cells are exposed to saidhypoxic condition for less than one hour, or less than thirty minutes,or is not exposed to a hypoxic condition, during collection, enrichmentor isolation. In another specific embodiment, said placental stem cellsare not exposed to shear stress during collection, enrichment orisolation.

The enhanced placental stem cells, as well as the placental stem cellsto be used to produce enhanced placental stem cells, described hereincan be cryopreserved, e.g., in cryopreservation medium in smallcontainers, e.g., ampoules. Suitable cryopreservation medium includes,but is not limited to, culture medium including, e.g., growth medium, orcell freezing medium, for example commercially available cell freezingmedium, e.g., C2695, C2639 or C6039 (Sigma). Cryopreservation mediumpreferably comprises DMSO (dimethylsulfoxide), at a concentration of,e.g., about 10% (v/v). Cryopreservation medium may comprise additionalagents, for example, Plasmalyte, methylcellulose with or withoutglycerol. The stem cells are preferably cooled at about 1° C./min duringcryopreservation. A preferred cryopreservation temperature is about −80°C. to about −180° C., preferably about −125° C. to about −140° C.Cryopreserved cells can be transferred to liquid nitrogen prior tothawing for use. In some embodiments, for example, once the ampouleshave reached about −90° C., they are transferred to a liquid nitrogenstorage area. Cryopreserved cells preferably are thawed at a temperatureof about 25° C. to about 40° C., preferably to a temperature of about37° C. In certain embodiments, enhanced placental stem cells providedherein are cryopreserved about 12, 24, 36, 48, 60 or 72 hours afterbeing contacted with modulatory RNA molecules (e.g., transfection). Inone embodiment, enhanced placental stem cells provided herein arecryopreserved about 24 hours after being contacted with modulatory RNAmolecules (e.g., transfection).

5.7 Compositions

5.7.1 Compositions Comprising Enhanced Placental Stem Cells

Provided herein are compositions comprising the enhanced placental stemcells described herein. Such compositions may comprise populations ofenhanced placental stem cells provided herein combined with anyphysiologically-acceptable or medically-acceptable compound, compositionor device for use in, e.g., research or therapeutics.

Enhanced placental stem cells can be prepared in a form that is easilyadministrable to an individual. For example, enhanced placental stemcells described herein can be contained within a container that issuitable for medical use. Such a container can be, for example, asterile plastic bag, flask, jar, vial, or other container from which theplacental cell population can be easily dispensed. For example, thecontainer can be a blood bag or other plastic, medically-acceptable bagsuitable for the intravenous administration of a liquid to a recipient.The container is preferably one that allows for cryopreservation of theenhanced placental stem cells.

Enhanced placental stem cell populations, e.g., cryopreserved enhancedplacental stem cell populations, can comprise enhanced placental stemcells derived from a single donor, or from multiple donors. The enhancedplacental stem cells can be completely HLA-matched to an intendedrecipient, or partially or completely HLA-mismatched.

Thus, in one embodiment, provided herein is a composition comprisingenhanced placental stem cells in a container. In a specific embodiment,the enhanced placental stem cells cryopreserved. In another specificembodiment, the container is a bag, flask, vial or jar. In more specificembodiment, said bag is a sterile plastic bag. In a more specificembodiment, said bag is suitable for, allows or facilitates intravenousadministration of said enhanced placental stem cells. The bag cancomprise multiple lumens or compartments that are interconnected toallow mixing of the enhanced placental stem cells and one or more othersolutions, e.g., a drug, prior to, or during, administration. In anotherspecific embodiment, the composition comprises one or more compoundsthat facilitate cryopreservation of the combined stem cell population.In another specific embodiment, said enhanced placental stem cells arecontained within a physiologically-acceptable aqueous solution. In amore specific embodiment, said physiologically-acceptable aqueoussolution is a 0.9% NaCl solution. In another specific embodiment, saidenhanced placental stem cells are HLA-matched to a recipient of saidenhanced placental stem cells. In another specific embodiment, saidenhanced placental stem cells are at least partially HLA-mismatched to arecipient of said enhanced placental stem cells. In another specificembodiment, said enhanced placental stem cells are derived fromplacental stem cells from a plurality of donors.

5.7.1.1 Pharmaceutical Compositions

In another aspect, provided herein is a pharmaceutical composition, saidpharmaceutical composition comprising a therapeutically effective amountof enhanced placental stem cells.

The enhanced placental stem cells provided herein can be formulated intopharmaceutical compositions for use in vivo. Such pharmaceuticalcompositions can comprise enhanced placental stem cells in apharmaceutically-acceptable carrier, e.g., a saline solution or otheraccepted physiologically-acceptable solution for in vivo administration.Pharmaceutical compositions provided herein can comprise any of theenhanced placental stem cells described herein. The pharmaceuticalcompositions can comprise fetal, maternal, or both fetal and maternalenhanced placental stem cells. The pharmaceutical compositions providedherein can further comprise enhanced placental stem cells produced fromplacental stem cells obtained from a single individual or placenta, orfrom a plurality of individuals or placentae.

The pharmaceutical compositions provided herein can comprise any numberof enhanced placental stem cells. For example, a single unit dose ofenhanced placental stem cells can comprise, in various embodiments,about, at least, or no more than 1×10⁵, 5×10⁵, 1×10⁶, 5×10⁶, 1×10⁷,5×10⁷, 1×10⁸, 5×10⁸, 1×10⁹, 5×10⁹, 1×10¹⁰, 5×10¹⁰, 1×10¹¹ or moreenhanced placental stem cells.

The pharmaceutical compositions provided herein can comprise populationsof enhanced placental stem cells that comprise 50% viable enhancedplacental stem cells or more (that is, at least 50% of the cells in thepopulation are functional or living). Preferably, at least 60% of thecells in the population are viable. More preferably, at least 70%, 80%,90%, 95%, or 99% of the enhanced placental stem cells in the populationin the pharmaceutical composition are viable.

5.7.1.2 Matrices Comprising Enhanced Placental Stem Cells

Further provided herein are matrices, hydrogels, scaffolds, and the likethat comprise enhanced placental stem cells. The enhanced placental stemcells provided herein can be seeded onto a natural matrix, e.g., aplacental biomaterial such as an amniotic membrane material. Such anamniotic membrane material can be, e.g., amniotic membrane dissecteddirectly from a mammalian placenta; fixed or heat-treated amnioticmembrane, substantially dry (i.e., <20%, <15%, <10%, <5%, <2%, or <1%H₂O) amniotic membrane, chorionic membrane, substantially dry chorionicmembrane, substantially dry amniotic and chorionic membrane, and thelike. Preferred placental biomaterials on which enhanced placental stemcells can be seeded are described in U.S. Application Publication No.2004/0048796.

The enhanced placental stem cells provided herein can be suspended in ahydrogel solution suitable for, e.g., injection. Suitable hydrogels forsuch compositions include self-assembling peptides, such as RAD16.Enhanced placental stem cells can also be combined with, e.g., alginateor platelet-rich plasma, or other fibrin-containing matrices, for localinjection. In one embodiment, a hydrogel solution comprising enhancedplacental stem cells can be allowed to harden, for instance in a mold,to form a matrix having the cells dispersed therein for implantation.Enhanced placental stem cells in such a matrix can also be cultured sothat the cells are mitotically expanded prior to implantation. Thehydrogel can be, e.g., an organic polymer (natural or synthetic) that iscross-linked via covalent, ionic, or hydrogen bonds to create athree-dimensional open-lattice structure that entraps water molecules toform a gel. Hydrogel-forming materials include polysaccharides such asalginate and salts thereof, peptides, polyphosphazines, andpolyacrylates, which are crosslinked ionically, or block polymers suchas polyethylene oxide-polypropylene glycol block copolymers which arecrosslinked by temperature or pH, respectively. In some embodiments, thehydrogel or matrix is biodegradable.

In some embodiments, the matrix comprises an in situ polymerizable gel(see., e.g., U.S. Patent Application Publication 2002/0022676; Anseth etal., J. Control Release, 78(1-3):199-209 (2002); Wang et al.,Biomaterials, 24(22):3969-80 (2003).

In some embodiments, the polymers are at least partially soluble inaqueous solutions, such as water, buffered salt solutions, or aqueousalcohol solutions, that have charged side groups, or a monovalent ionicsalt thereof. Examples of polymers having acidic side groups that can bereacted with cations are poly(phosphazenes), poly(acrylic acids),poly(methacrylic acids), copolymers of acrylic acid and methacrylicacid, poly(vinyl acetate), and sulfonated polymers, such as sulfonatedpolystyrene. Copolymers having acidic side groups formed by reaction ofacrylic or methacrylic acid and vinyl ether monomers or polymers canalso be used. Examples of acidic groups are carboxylic acid groups,sulfonic acid groups, halogenated (preferably fluorinated) alcoholgroups, phenolic OH groups, and acidic OH groups.

The enhanced placental stem cells can be seeded onto a three-dimensionalframework or scaffold and implanted in vivo. Such a framework can beimplanted in combination with any one or more growth factors, cells,drugs or other components that stimulate tissue formation or otherwiseenhance or improve the practice of the methods of treatment describedelsewhere herein.

Examples of scaffolds that can be used herein include nonwoven mats,porous foams, or self assembling peptides. Nonwoven mats can be formedusing fibers comprised of a synthetic absorbable copolymer of glycolicand lactic acids (e.g., PGA/PLA) (VICRYL, Ethicon, Inc., Somerville,N.J.). Foams, composed of, e.g., poly(ε-caprolactone)/poly(glycolicacid) (PCL/PGA) copolymer, formed by processes such as freeze-drying, orlyophilization (see, e.g., U.S. Pat. No. 6,355,699), can also be used asscaffolds.

In another embodiment, the scaffold is, or comprises, a nanofibrousscaffold, e.g., an electrospun nanofibrous scaffold. In a more specificembodiment, said nanofibrous scaffold comprises poly(L-lactic acid)(PLLA), type I collagen, a copolymer of vinylidene fluoride andtrifluoroethylnee (PVDF-TrFE), poly(-caprolactone),poly(L-lactide-co-ε-caprolactone) [P(LLA-CL)] (e.g., 75:25), and/or acopolymer of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) andtype I collagen. Methods of producing nanofibrous scaffolds, e.g.,electrospun nanofibrous scaffolds, are known in the art. See, e.g., Xuet al., Tissue Engineering 10(7):1160-1168 (2004); Xu et al.,Biomaterials 25:877-886 (20040; Meng et al., J. Biomaterials Sci.,Polymer Edition 18(1):81-94 (2007).

The enhanced placental stem cells described herein can also be seededonto, or contacted with, a physiologically-acceptable ceramic materialincluding, but not limited to, mono-, di-, tri-, alpha-tri-, beta-tri-,and tetra-calcium phosphate, hydroxyapatite, fluoroapatites, calciumsulfates, calcium fluorides, calcium oxides, calcium carbonates,magnesium calcium phosphates, biologically active glasses such asBIOGLASS®, and mixtures thereof. Porous biocompatible ceramic materialscurrently commercially available include SURGIBONE® (CanMedica Corp.,Canada), ENDOBON® (Merck Biomaterial France, France), CEROS® (Mathys,AG, Bettlach, Switzerland), and mineralized collagen bone graftingproducts such as HEALOS™ (DePuy, Inc., Raynham, Mass.) and VITOSS®,RHAKOSS™, and CORTOSS® (Orthovita, Malvern, Pa.). The framework can be amixture, blend or composite of natural and/or synthetic materials.

In another embodiment, enhanced placental stem cells can be seeded onto,or contacted with, a felt, which can be, e.g., composed of amultifilament yarn made from a bioabsorbable material such as PGA, PLA,PCL copolymers or blends, or hyaluronic acid.

The enhanced placental stem cells described herein can, in anotherembodiment, be seeded onto foam scaffolds that may be compositestructures. Such foam scaffolds can be molded into a useful shape. Insome embodiments, the framework is treated, e.g., with 0.1M acetic acidfollowed by incubation in polylysine, PBS, and/or collagen, prior toinoculation of the enhanced placental stem cells in order to enhancecell attachment. External surfaces of a matrix may be modified toimprove the attachment or growth of cells and differentiation of tissue,such as by plasma-coating the matrix, or addition of one or moreproteins (e.g., collagens, elastic fibers, reticular fibers),glycoproteins, glycosaminoglycans (e.g., heparin sulfate,chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratinsulfate, etc.), a cellular matrix, and/or other materials such as, butnot limited to, gelatin, alginates, agar, agarose, and plant gums, andthe like.

In some embodiments, the scaffold comprises, or is treated with,materials that render it non-thrombogenic. These treatments andmaterials may also promote and sustain endothelial growth, migration,and extracellular matrix deposition. Examples of these materials andtreatments include but are not limited to natural materials such asbasement membrane proteins such as laminin and Type IV collagen,synthetic materials such as EPTFE, and segmented polyurethaneureasilicones, such as PURSPAN™ (The Polymer Technology Group, Inc.,Berkeley, Calif.). The scaffold can also comprise anti-thrombotic agentssuch as heparin; the scaffolds can also be treated to alter the surfacecharge (e.g., coating with plasma) prior to seeding with enhancedplacental stem cells.

6. EXAMPLES

6.1 Example 1: Identification of Survival-Associated Genes Associatedwith Placental Stem Cell Survival

This example describes the identification of molecular pathways andspecific genes associated with the response of placental stem cells toenvironmental insult. Increases of cellular caspase 3/7 activity as wellas decreases in cellular metabolic activity and mitochondrial membranepotential are known to be associated with decrease in cellular survival.Accordingly, augmentation of survival was assessed by measurement ofcaspase 3/7 activity, metabolic activity, and mitochondrial membranepotential of placental stem cells following insult.

6.1.1 Methods

6.1.1.1 MicroRNA Library Screen

A microRNA library screen was used to identify microRNA capable ofaugmenting the survival of placental stem cells exposed to environmentalinsult. Placental stem cells were seeded on tissue culture plates for 24hours, transfected with a microRNA library for 24 hours, then insultedwith 100% normal rat serum overnight. Following the overnight culture ininsult (100% rat serum), the cells were assayed for cell survivalaugmentation. The protocol is described in greater detail below.

6.1.1.1.1 MicroRNA Transfection

CD34⁻, CD10⁺, CD105⁺, CD200⁺ Placental stem cells were seeded in 96-welltissue culture plates, in duplicate, at a concentration of 3×10³ (“Day0”). After 24 h, on Day 1, the cells were transfected with 100 nM of theAmbion Pre-miR Mimic Library (miRBase v15), appropriate controls (notreatment; vehicle; and negative controls 1 and 2, obtained from themanufacturer), and Ambion's Amine transfection reagent.

6.1.1.1.2 Environmental Insult

On Day 2, the transfection media was removed and replaced with eitherculture medium or 100% Normal Rat Serum (insult) (Invitrogen) andincubated overnight.

6.1.1.1.3 Insult/Capase 3/7 Assessment

On Day 3 the insult media was removed, replaced with 100 μL of 0.5 μMHoechst 33342 stain, and the cells were incubated for 1 hour at 37° C.The stained cells were then imaged using an InCell Analyzer (GE) andnuclei counts were analyzed. After imaging, the Hoechst stain wasremoved and the cells were incubated with 100 μL of Caspase-Glo 3/7(Promega) reagent for 1 hour at room temperature. Following incubation,absolute luminescence was read using a Biotek Synergy Plate Reader, andLuminescence readouts and nuclei counts were used to calculate caspase3/7 activity/cell. Hits were analyzed against both negative controls andmicroRNAs that significantly lowered caspase 3/7 activity in theplacental stem cells vs. either Negative Control (p<0.05, Student'st-Test) were identified.

6.1.1.2 Metabolic Activity and Mitochondrial Membrane Potential Studies

In addition to the microRNA library screen, metabolic activity andmitochondrial membrane potential of placental stem cells exposed toenvironmental insult was assessed using the Cell Titer Glo Assay(Promega) and the TMRE Mitochondrial Membrane Potential Assay Kit(Abcam), respectively.

For the assessment of metabolic activity, placental stem cells exposedto insult (rat serum) as described above were incubated with 100 μL ofCell Titer Glo (Promega) reagent for 15 minutes at room temperature.Following incubation, absolute luminescence was read using a BiotekSynergy Plate Reader, and luminescence readouts and nuclei counts wereused to calculate metabolic activity/cell.

For the assessment of mitochondrial membrane potential, placental stemcells exposed to insult (rat serum) as described above were incubatedwith 1.5 μM of TMRE stain (Abcam) reagent for 30 minutes at 37° C.Following incubation, cells were washed twice with 0.2% BSA-PBS, andfluorescence was read using a Biotek Synergy Plate Reader. The meanfluorescence intensity readouts and nuclei counts were used to calculatemitochondrial membrane potential/cell.

For both assays, hits were analyzed against both negative controls(Negative Control #1 and Negative Control #1, obtained from Ambion) andidentified as microRNAs that significantly improved placental stem cellmetabolic activity/cell and/or mitochondrial membrane potential/cell vs.either negative control (p<0.05, Student's t-Test).

6.1.1.3 Bioinformatic Target Gene Prediction/Pathway Analysis

A database consolidating of ten microRNA prediction databases wasutilized to generate the predicted target genes of the confirmedmicroRNA hits from the library screen. This database consisted ofprediction algorithms from Diana-microT, miRDB, miRTar, miRanda,miRBase, picTar, Targetscan, TarBase, miRecords, and miRTarBase. Thepathway analysis and biological function information of predicted targetgenes were completed using Ingenuity Pathway Analysis (IPA) (IngenuitySystems).

6.1.1.4 MicroRNA and Target Gene Validation Studies with PCR

For MicroRNA/target gene validation studies, placental stem cells weretransfected with microRNA library screen confirmed hits, as describedabove but without insult, and microRNA and mRNA were isolated using theMiRvana microRNA Isolation Kit (Ambion). Serum starvation (culture inDMEM) and contact inhibition (doubling the cell seeding number) wereconditions included in the study as positive cell cycle arrest controlsto compare with microRNA gene modification.

For microRNA transfection assessment, RT-PCR was completed using TaqmanMicroRNA Reverse Transcription Kit and Taqman MicroRNA Assays (LifeTechnologies) specific for each transfected microRNA mimic. PCR wascompleted using the Taqman MicroRNA Assays with Taqman 2× Universal PCRMaster Mix with No AMPErase UNG (Life Technologies) and cDNA, and wasrun in standard mode on the Applied Biosystems 7900HT Fast Real-Time PCRInstrument. All data was analyzed in RQ Manager (Applied Biosystems).

To assess mRNA/target gene expression, RT-PCR was completed using theSuperscript VILO Master Mix (Life Technologies). PCR was completed usingTaqman Gene Expression Assays (Life Technologies) specific for predictedtarget genes, Taqman Fast Universal PCR Master Mix (Life Technologies),and cDNA, and was run on the Applied Biosystems 7900HT Fast Real-TimePCR Instrument in fast mode. All data was analyzed in RQ Manager(Applied Biosystems).

6.1.1.5 MicroRNA Cell Cycle Validation

A microRNA cell cycle validation study was completed to test whethermicroRNA cell survival hits induce a state of cellular quiescence inplacental stem cells that protects the cells against insult.

Placental stem cells were seeded and transfected with microRNA cellsurvival hits as described above, but without 100% Rat Serum insult.Cells were then harvested and stained with the BrdU Flow Kit (BDPharmingen), as per manufacturer's protocol, for cell cycle distributionanalysis.

In addition, placental stem cells were evaluated for the effects of themicroRNA cell survival hits on the expression of key cell cycleproteins, using antibodies against human CDK6, Cyclin D1 (CCND1), CyclinD3 (CCND3), and Cyclin E (CCNE1) proteins. Standard techniques were usedfor CDK6 and CCND1.

For Cyclin D3 staining, cells were fixed with 1% Formaldehyde for 15minutes at 4° C., followed by incubation with cold 75% Ethanol for aminimum of 2 hours at −20° C. Cells were permeabilized with cold 0.25%Triton X-100 for 5 minutes at 4° C., washed with 1% FBS-PBS, andincubated with FcR Blocking Reagent (Miltenyi Biotec) for 10 minutes at4° C. Placental stem cells were then stained with 2.5× manufacturer'srecommended staining concentration of Mouse anti-Human Cyclin D3 Fitcantibody (BD Pharmingen), with matched concentration of Mouse IgG1Isotype control (BD Pharmingen), and stained for 30 minutes at roomtemperature and in the dark. Data was acquired using a FACS Canto IIFlow Cytometer (BD Biosciences) and analyzed in FlowJo (Tree Star).

For Cyclin E staining, cells were fixed with 4% Paraformaldehyde for 15minutes at 4° C., and permeabilized with cold 0.1% Tween-20 for 20minutes at 4° C. Cells were washed with 1% FBS-PBS, and incubated withFcR Blocking Reagent (Miltenyi Biotec) for 10 minutes at 4° C. Placentalstem cells were then stained for 30 minutes at room temperature with2.5× manufacturer's recommended staining concentration of Mouseanti-Human Cyclin E antibody (Abcam), with matched concentration ofMouse IgG1 Isotype control (Abcam), followed by secondary staining withGoat Polyclonal Secondary to Mouse IgG Dylight 488(Abcam) for 30 minutesat room temperature. Data was acquired using a FACS Canto II FlowCytometer (BD Biosciences) and analyzed in FlowJo (Tree Star).

6.1.2 Results

6.1.2.1 MicroRNA Library Screen

The screening of 1,090 microRNAs for augmentation of placental stem cellsurvival upon exposure to 100% Rat Serum insult resulted in diverseeffects caspase 3/7 activity/cell in placental stem cells.

Several transfected microRNAs lowered caspase 3/7 activity/cellpost-insult. As shown in Table 3, a total of 36 microRNA hitssignificantly decreased caspase 3/7 activity/cell post insult, ascompared to either of the negative controls provided in the assay kit.Modulation ranged from a decrease of −7.23% to −34.44% in caspase 3/7activity/cell.

TABLE 3 List microRNA hits from the library screen for placental stemcell survival augmentation MicroRNA Library Screen for PDAC CellSurvival Augmentation Hits hsa-miR-424 hsa-miR-141 hsa-miR-3142hsa-miR-4310 hsa-miR-1826 hsa-miR-1308 hsa-miR-143 hsa-miR-581hsa-miR-1201 hsa-miR-1203 hsa-miR-3158 hsa-miR-1227 hsa-miR-136hsa-miR-362-5p hsa-miR-4305 hsa-miR-1271 hsa-miR-1236 hsa-miR-369-5phsa-miR-662 hsa-miR-613 hsa-miR-126* hsa-miR-3123 hsa-miR-432hsa-miR-450b-5p hsa-miR-432* hsa-miR-611 hsa-miR-591 hsa-miR-631hsa-miR-3170 hsa-miR-548k hsa-miR-16 hsa-miR-199a-5p hsa-miR-521hsa-miR-301a hsa-miR-514b-5p hsa-miR-29a

6.1.2.2 Metabolic Activity and Mitochondrial Membrane Potential Studies

Each of microRNA MiR-16, miR-29a, miR-424, miR-4305, miR-3142, andmiR-613 were determined to cause a statistically significant decrease incaspase 3/7 activity/cell post insult as compared to the negativecontrols (FIG. 1).

Each of these microRNA hits was also found to increase placental stemcell metabolic activity/cell post insult (FIG. 2). Of the six confirmedhits, miR-16 and miR-424 also increased placental stem cellmitochondrial membrane potential/cell (FIG. 3), further confirming cellsurvival augmentation.

The top confirmed caspase 3/7 modulators, miR-16, miR-424, and miR-29a,were found to share similar seed sequences that bind to their targetmRNA, i.e., the 5′ sequence of miRNA important to function of the miRNA(based on its complementarity to the nucleic acid sequence of the targetmRNA). MiR-16 and miR-424 have identical seed sequences, while miR-29avaries by one nucleotide in the 5th position (Table 4). Accordingly, itwas expected that these microRNA target and modulate a similar subset oftarget genes and pathways/biological functions.

TABLE 4 Seed sequence similarity between microRNA (seed sequences aspresent in the mature miRNA are boxed) Confirmed Cell Survival miR HitsmiRNA Mature Confirmation Screens: Name(s) Ambion_miProd_IDMature_Sequence Change in Caspase Activity hsa-miR-16 PM10339

21-23% Decrease hsa-miR-424 PM10306

12-14% Decrease hsa-miR-29a PM12499

17-20% Decrease hsa-miR-613 PM11528 AGGAAUGUUCCUUCUUUGCC 4% Decreasehsa-miR-3142 PM18673 AAGGCCUUUCUGAACCUUCAGA 6% Decrease hsa-miR-4305PM18703 CCUAGACACCUCCAGUUC 8% Decrease

6.1.2.3 Target Gene Prediction/Pathway Analysis

Bioinformatic analysis of miR-29a, miR-16, and miR-424 identified atotal of 150 experimentally validated genes that were predicted to betargeted by these microRNAs. Prediction analysis of miR-29a resulted in26 target genes, analysis of miR-16 predicted 105 target genes, whileanalysis of miR-424 resulted in 19 genes (Table 5).

TABLE 5 MicroRNA target gene prediction of experimentally validatedgenes for miR-29a, miR-16, and miR-424 Experimentally Validated Genes(Number in parentheses denotes # of predicted databases) hsa-miR-29ahsa-miR-16 hsa-miR-424 ADAMTS9 (5) ABCF2 (2) DNAJB4 (3) MYB (5) RTN4 (2)ANLN (1) BACE1 (3) ABHD10 (1) EGFR (1) NAA15 (3) SEC24A (1) CCND1 (3)BCL2 (1) ACTR1A (2) EIF4E (2) NAA25 (2) SHOC2 (4) CCND3 (2) CAV2 (3)ACVR2A (5) EPT1 (3) NAPG (2) SLC12A2 (4) CCNE1 (5) CD276 (3) ADSS (1)FGF2 (3) NOB1 (2) SLC16A3 (1) CCNF (1) CDC42 (2) ALG3 (1) FNDC3B (2)NOTCH2 (2) SLC25A22 (1) CDC14A (1) CDK6 (3) ARHGDIA (3) GALNT7 (1)PAFAH1B2 (3) SLC38A5 (1) CDC25A (5) COL3A1 (5) ARL2 (3) GPAM (1) PDCD4(4) SLC7A1 (1) CDK6 (2) COL4A1 (4) ATG9A (3) HACE1 (1) PDCD6IP (2) SNX15(1) CHEK1 (4) COL4A2 (2) BCL2 (3) HARS (1) PHKB (1) SPTLC1 (3) CUL2 (7)CPEB3 (2) C9ORF167 (1) HARS2 (1) PISD (2) SQSTM1 (1) FGFR1 (6) CXXC6 (1)C9ORF89 (2) HERC6 (1) PLK1 (1) SRPR (5) ITPR1 (6) DIABLO (1) CACNA2D1(1) HMGA1 (4) PNN (1) SRPRB (2) KIF23 (5) DNMT3A (5) CAPRIN1 (2) HSDL2(1) PNPLA6 (3) TMEM43 (1) MAP2K1 (5) DNMT3B (3) CCDC109A (1) IGF2R (2)PPIF (1) TNFSF9 (1) MYB (7) FGA (1) CCND1 (3) IPO4 (1) PPM1D (4) TOMM34(2) PIAS1 (1) IMPDH1 (2) CCND3 (2) ITGA2 (3) PPP2R5C (3) TPM3 (2) PLAG1(5) INSIG1 (3) CCNE1 (5) KCNN4 (3) PSAT1 (2) TPPP3 (2) SIAH1 (5) KREMEN2(2) CCNT2 (6) KPNA3 (2) PTCD3 (1) UBE2V1 (1) WEE1 (6) LPL (3) CDC14B (2)LAMC1 (1) PTGS2 (1) UBE4A (1) MCL1 (1) CDK5RAP1 (1) LAMTOR3 (1) PURA (4)UGDH (1) PIK3R1 (4) CDK6 (1) LUZP1 (3) RAB9B (2) UTP15 (1) PPM1D (4)CENPJ (1) LYPLA2 (2) RAD51C (1) VEGFA (4) SPARC (3) CHORDC1 (3) MCL1 (1)RARS (1) WNT3A (5) TET1 (4) CREBL2 (1) MLLT1 (1) RECK (5) WT1 (2) TRIM63(3) CSHL1 (1) MMS19 (2) RNASEL (1) YIF1B (1) ZNF622 (3) 26 105 19 150TOTAL

Several of the experimentally validated genes were predicted to betargeted by two or more microRNA hits (Tables 5 and 6). These predictedgenes included genes related to apoptosis, cell cycle regulation,transcription activation, and the cell stress response pathway.

TABLE 6 List and function of experimentally validated genes predicted tobe targeted by two or more confirmed placental stem cell survivalaugmenting microRNA hits Genes targeted by >2 miRs (Number inparentheses denotes # of mirs) miR-29a, BCL2 (2) B-cell lymphoma 2;Apoptosis Regulator: miR-16 Anti-apoptotic; Implicated in a number ofCancers miR-16, CCND1 (2) Cyclin D1; Forms a complex with miR-424 CDK4and CDK6 and initiates G1/S phase cell cycle transition; Over expressedin a variety of tumors miR-16, CCND3 (2) Cyclin D3; Forms a complex withCDK4 miR-424 and CDK6 and initiates G1/S phase cell cycle transitionmiR-16, CCNE1 (2) Cyclin E1; Forms a complex with CDK2 and miR-424initiates G1/S phase cell cycle transition; Over expressed in a varietyof tumors miR-29a, CDK6 (3) Cyclin-Dependent Kinase 6; AssociatesmiR-16, with Cyclin D to initiate G1/S phase miR-424 cell cycletransition/progression miR-29a, MCL1 (2) Induced Myeloid Leukemia CellDifferentiation miR-16 Protein; Alternative splicing of gene can resultin a gene product that either inhibits or promotes apoptosis miR-16, MYB(2) Myeloblastosis Proto-Oncogene Protein/ miR-424 TranscriptionalActivator; Transcription Factor; Plays a role in the regulation ofhematopoiesis and tumorigenesis miR-29a, PPM1D (2) Protein phosphatase1D; Ser/Thr protein miR-16 phosphatase; Negative regulator of cellstress response pathways; Reduces p53-mediated transcription and stressinduced apoptosis; Plays a role in Cancer development

6.1.2.4 MicroRNA and Target Gene Validation Studies with PCR

MicroRNA validation studies using qPCR confirmed successful transfectionof placental stem cells with microRNA mimics prior to insult.Transfection of placental stem cells with miR-29a resulted in a2.78-7.47 fold increase in miR-29a expression (FIG. 4A), transfectionwith miR-16 resulted in a 762-2,158 fold increase in miR-16 expression(FIG. 4B), and transfection with miR-424 resulted in a 274-914 foldincrease in miR-424 expression (FIG. 4C).

Gene expression studies validated and confirmed numerous genes predictedto be targeted by the microRNA hits in the bioinformatic studies. All ofthe experimentally validated genes that were predicted to be targeted bytwo or more microRNA hits (italicized) were confirmed to be targeted andmodulated by the microRNA hits within the gene expression studies.

miR-16 and miR424 had similar target gene modulation patterns that wereconsistent with serum starvation and contact inhibition conditionsignatures, but distinct from miR-29a. MiR-16 and miR-424 bothdown-regulated genes related to cell cycle progression (CCND1, CCND3,CCNE1, CCNF, and CDK6), cell cycle regulation (CDC25A, WEE1, and CHEK1)and transcription activation (MyB). MiR-16 and miR-424 up-regulatednegative regulators of cell growth and division (PPP2R5C), inhibitors ofapoptosis (MCL1), negative regulators of cell stress pathway/p53 (PPMIDand HMGA1), as well as genes related to cell survival signaling (AKT3and VEGFA) and adhesion (ITGA2). These molecular changes, taken togetherand compared to changes resulting from serum starvation and contactinhibition, suggest a role of cell cycle arrest as a mechanism forplacental stem cell survival augmentation with miR-16 and miR-424.

miR-29a up-regulated CCND1, CCND3, CCNE1, CDC25A, WEE1 gene expression,and down-regulated MCL1, PPMID, HMGA1, AKT3, VEGFA, and ITGA2 geneexpression, implicating a slightly different underlying mechanism forcell survival augmentation for this microRNA.

The molecular changes observed in the target gene validation studiesindicate that these miR treatments induce a state of quiescence inplacental stem cells that is protective against insult, and augmentscell survival.

6.1.2.5 MicroRNA Cell Cycle Validation

Placental stem cell microRNA cell cycle validation studies, using BrdU,confirmed that the microRNA cell survival hits described above induce astate of cellular quiescence in placental stem cells that is protectiveagainst insult. Transfection of placental stem cells with miR-29aresulted in a 3.32% to 4.77% absolute increase in the distribution ofcells in the G0/G1-phase (FIG. 5A), a −1.4% to −3.32% absolute decreasein the distribution of cells in the S-phase (FIG. 5B), and a −1.8% to−2.05% absolute decrease in the distribution of cells in the G2/M-phaseof the cell cycle (FIG. 5C). Transfection with miR-16 resulted in a15.45% to 16.9% absolute increase in the distribution of cells in theG0/G1-phase (FIG. 5A), a −3.27% to −5.2% absolute decrease in thedistribution of cells in the S-phase (FIG. 5B), and a −9.99% to −10.24%absolute decrease in the distribution of cells in the G2/M-phase of thecell cycle (FIG. 5C). Transfection with miR-424 resulted in a 11.85% to13.3% absolute increase in the distribution of cells in the G0/G1-phase(FIG. 5A), a −2.88% to −4.81% absolute decrease in the distribution ofcells in the S-phase (FIG. 5B), and a −8.27% to −8.52% absolute decreasein the distribution of cells in the G2/M-phase of the cell cycle (FIG.5C).

Flow cytometric evaluation further validated Cyclin D3 and Cyclin E astargets of miR-16 and miR-424. Transfection with miR-16 resulted in a−29.55% to −35.25% absolute decrease in placental stem cell Cyclin D3expression (FIG. 6A), and a −20.75% to −23.2% absolute decrease inplacental stem cell Cyclin E expression (FIG. 6B). Transfection withmiR-424 resulted in a −20.25% to −25.95% absolute decrease in placentalstem cell Cyclin D3 expression (FIG. 6A), and a −11.15% to −13.6%absolute decrease in placental stem cell Cyclin E expression (FIG. 6B).

Transfection of placental stem cells with miR-29a resulted in anup-regulation of Cyclin D3 and Cyclin E gene expression by the placentalstem cells, as well as an increase in Cyclin D3 and Cyclin E proteinexpression (FIG. 6A-B). Without wishing to be bound by any particulartheory or mechanism, based on the cell cycle studies described above,cell cycle arrest is the most likely mechanism of placental stem cellsurvival augmentation with miR-29a. However, these data indicate thatthe mechanism is not likely mediated by the targeting anddown-regulation of Cyclins D3 and E, as in the case of miR-16 andmiR-424.

These cell cycle validation studies further demonstrate that modulatoryRNA, such as microRNA, can augment placental stem cell survival in theface of insult by targeting cell cycle related genes/proteins.

6.1.3 Conclusion

microRNAs capable of augmenting placental stem cell survival in thepresence of insult were identified and validated, as were the moleculartargets and biological pathways/mechanisms linked to cell survivalaugmentation. The data indicate that targeting of the molecular targetsand biological pathways/mechanisms results in induction of quiescence inthe placental stem cells with enhanced survival capability. Modulationof microRNAs and their targeted genes/pathways (e.g., through mechanismsother than use of microRNA) represents a novel means by which enhancedplacental stem cells capable of surviving under suboptimal conditions,or surviving for longer durations of time under normal culture/in vivoconditions, can be generated.

Equivalents

The compositions and methods disclosed herein are not to be limited inscope by the specific embodiments described herein. Indeed, variousmodifications of the compositions and methods in addition to thosedescribed will become apparent to those skilled in the art from theforegoing description and accompanying figures. Such modifications areintended to fall within the scope of the appended claims.

Various publications, patents and patent applications are cited herein,the disclosures of which are incorporated by reference in theirentireties.

What is claimed is:
 1. An isolated placental stem cell, wherein said placental stem cell has been modified to comprise or express miR-29a, and wherein said placental stem cell demonstrates increased survival relative to corresponding unmodified placental stem cells when cultured under one or more conditions that cause cell death.
 2. The isolated placental stem cell of claim 1, wherein said placental stem cell expresses at least one survival-associated gene at a decreased level as compared to the expression of the same survival-associated gene in a corresponding unmodified placental stem cell.
 3. The isolated placental stem cell of claim 2, wherein said survival-associated gene is ADAMTS9, BACE1, BCL2, CAV2, CD276, CDC42, CDK6, COL3A1, COL4A1, COL4A2, CPEB3, TRIM63, CXXC6/TET1, DIABLO, DNMT3A, DNMT3B, FGA, IMPDH1, INSIG1, KREMEN2, LPL, MCL1, PIK3R1, PPM1D, SPARC.
 4. The isolated placental stem cell of claim 1, wherein said miR-29a causes said placental cell to express Cyclin D3 and/or Cyclin E at an increased level as compared to a corresponding unmodified placental stem cell.
 5. The isolated placental stem cell of claim 1, wherein said placental stem cell exhibits (i) decreased expression of caspase 3/7, (ii) increased mitochondrial membrane potential, and/or (iii) increased metabolic activity when cultured under one or more conditions that cause cell death as compared to corresponding unmodified placental stem cells cultured under the same condition(s).
 6. An isolated population of cells, wherein at least 50% of the cells in said population are the cells of claim
 1. 7. The isolated population of cells of claim 6, wherein at least 80% of the cells in said population of cells are the cells of claim
 1. 8. The population of cells of claim 6, wherein said placental stem cells in said population are CD10⁺, CD34⁻, CD105⁺, CD200⁺placental stem cells.
 9. A composition comprising the isolated placental stem cell of claim
 1. 10. The placental stem cell of claim 1, wherein said placental stem cell is a CD10⁺, CD34⁻, CD105⁺, CD200⁺placental stem cell.
 11. The composition of claim 1, wherein said placental stem cells are CD10⁺, CD34⁻, CD105⁺, CD200⁺placental stem cells.
 12. A method of producing placental stem cells that comprise or express miR-29a, wherein said placental stem cells demonstrate increased survival relative to corresponding unmodified placental stem cells when cultured under one or more conditions that cause cell death, said method comprising contacting a population of placental stem cells with an effective amount of miR-29a, such that said placental stem cells, after having been contacted with said miR-29a express Cyclin D3 and/or Cyclin E at an increased level as compared to a corresponding number of unmodified placental stem cells not contacted with said miR-29a.
 13. An isolated placental stem cell or population thereof produced by the method of claim
 12. 14. A composition comprising an isolated placental stem cell produced by the method of claim
 12. 15. The method of claim 12, wherein said placental stem cells are CD10⁺, CD34⁻, CD105⁺, CD200⁺placental stem cells. 